Document 6524068

.
.A. Cover Sheet
FINAL REPORT
Covering Period: October 2000-March 2004
Submitted to the U.S. Agency for International Development: Bureau for Economic G r o u ~ .
.4piculture. and Trade
IlLPROVEMENT OF GROU'TH .AND SURVIVAL IN CULTURED RABBITFISH (SIG.4.Yl :T
GL'7T.4T L . 9
Principal Investigator: Dr. Bruria Funkenstein
Grantee Institution: Israel Oceanographic % Limnological Research
Tel-Shihona P.0.B 8030. Haifa 3 1080. Israel
Tel: 9724-8565233. e-mail: bruria~ocran.org.il
Collaborator: Prof. h i e h Genler
Institution: The Hebrew l'niversiv of Jerusalem
Collaborator: Dr. Felix G. Ayson
Institution: Southeast Asian Fisheries Development Center
Project Number: C 19-001
Grant Number: TA-MOU-99-C 19-00 1
Grant Project Officer: Mr. William Crane
Project Duration: April 1.2000-March 3 I. 2004
+
B . Table of Contents
.A . Cover Page ..............................................................
1
B . Table of Content ........................................................
2
C. Executive Summary....................................................
3
. .
D . Research Object~ves.................................................. -4
E . Methods and Results ................................................... 4
F. Impact. Relevance.and Technolog?. Transfer.......................6
G . Project .4ctivitiedOutputs............................................. 6
..
H . Project Product~ulty.................................................... 6
I . Future Work ...........................................................
.
-7
.
.A ppendis . Publ~caoonNo .1 ......................................... 8
Publicalion No . :!....................................... 17
Publication No. 3 ...................................... 27
i
C. Executive Summan.:
2
The purpose of this project was to improve the grouzh and sumival of the rabhitfish.
Sipnus guffuftu. cultured in Southeast Asia by underslanding the rate-limiting componenqs) in
the grouth hormone-insulin-like growth factor system. which might explain the slow -gonth m e
during the early juvenile stage and use this information to enhance p u t h rate by a d m i n i d o n
of groulh hormone using various methodologies.
During the project. grouth hormone (GH) from rabbitfish uas purified and antiserum \\as
raised in rabbits. The native protein purified at he
nart
of the project \\as used in p w z h
enhancement experiments of rabbitfish kept under various salinities. The cDK";As for rabbitfish
GH and insulin-like grouth factors I and II (IGF-I and IGF-11) were also cloned. Recombinant GH
protein was expressed in bacteria in large quantities and purified. The e f f e c t i v m of he
recombinant hormone to enhance grouth u ~ a stested in rirw by injecting he hormone to rabbitfish
and monitoring grouth (increases in body weight and body length). For testing he biological
activit!- in rirro. GH was added to liver pieces removed from rabbitfish and l
i
e for shon
incubation periods. Levels of IGF-I mRNA were determined. Experiments were also carried oul
(by using molecular biology methods) in u-hich he level of expression of two genes for p u t h
factors (IGF-I and IGF-11) was determined during earl>-stages of development of he &bitfish.
The results obtained suggest that administration of a little exogenous GH to he fr?- significantly
improved its g o n t h and rearing fr?. in dilute seawater likeuise improved its grouth performance.
The collaboration during the project included a visit of the Philippine PI to he Israeli Pls
laboratories. During the visit experiments were initiated to test various uals of introdwing
hormones into microdiet of fish Ian-ae. In addition the Philippine PI and he Israeli PI met during
the Fish Endocrinoloa Meeting and took this opportunip to discuss h e mul& of he project
and planning future work.
The funding provided by the project. enabled Dr. A y n to acquire imponant pieces of
equipment needed for the production of recombinant f f i H in his laboraton-. In addition. a
fermentor. freeze-drier and a big refrigerated centrifuge were provided by a pant from the
Japanese Gowrnment.
.
D. Research Obiectives:
4
1.
Purification of S. gullarus GH polypeptide and raising antibodies.
.7
Molecular cloning of S. prrlufzrs GH and IGF-I cDNAs.
3.
Cloning of GH and IGF-I in expression vectors for producing large
quantities.
4.
Effect of exogenous natitr S guttatus GH on growIII.
5.
Effect of exogenous bovine GH and low salinity on growIII.
6.
Production of large quantities of S prrranls recombinant GH polypeptide.
7.
Production of S. gutratus recombinant IGF-I poljpeptide.
8.
Assays to rest the biological activities of recombinant S. gutfarus GH.
9.
Extending the half-life of the recombinant S. gutratus GH.
E. Methods and Results:
I lsolarion o/-growthhormone lGH)from rabbi!fish
GH was emacted from p i t u i w glands under alkaline conditions. tiactionated by gel filtration
chromatography on Sephadex G-100. and purified b?; high performance liquid chromampphy
(The results are summarized in publication No. I J.
2. Cloning ofrahhi!fishGH cD.X4
A combination of RT-PCR and RACE (rapid amplification of cDNA ends) resulted in a cDN.4 of
860 nt. excluding the poly(A) tail. The unmslated regions are 94 and 175 bp in he 5' and 3'
ends. respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino
acids and a mature protein of 178 amino acid residues (The results are summarized in Publicarion
No. I ).
3 Cloning ojrubbiesh IGF-I cD.V.4
Rabbitfish IGF-I cDNA was successfully cloned from the liver. The preprolGF-l is composed of
186 amino acids (aa): this is composed of 4 aa for the signal peptide. 68 aa for he mature
peptide and 71 aa for the E domain. Only one form of Edomain is found in rabbitfish IGF-I (The
results see summarized in publication No. 2).
4. Cloning 01-rabbiifishIGF-II cD.Y4 (nor in the original proposall
Rabbitfish IGF-I1 cDNA was also successfully cloned from the liver. The preprolGF-ll is 215 aa
long: this is composed of 1 7 aa for the signal peptide. 70 aa for he mature peptide and 98 aa for
the E domain (The results are summarized in publication No. 2).
3. Drvelopnrenral espression o f IGFs in rubhir/ish
In order to examine if there is differential regulation of both lGFs during early development in
rabbitfish. primers were designed for both rabbitfish IGF-I and I1 and used it for RT-PCR of
.
3
samples from different stages of embnogenesis and different l m a l stages. IGF-II transcripl uas
e\pressed in unfertilized eggs (although \ e n ueak) and a \en. strong expression uas alrpad?
ohsened during morula stage (about 3 hours after fertil~zation)and this uas maintained until
hatching. On the other hand. IGF-I transcript \\as observed only in embpos near hatching. In he
lanae. ho\ve\-er. both lGFs u-ere more or less equally expressed (The results are summarized in
publication No. 2).
6 Erperinrenrs to enhance growrh in rabbi@sh
a ) Treatment with bovine GH to improve p u t h in rabbitfish 6y:
Rahbittish f n u-ere injected uith bovine GH at doses of 0.1 and 0.01 p@g BW. GH injection \\as
gken weekl!- for 4 weeks and the experiment lasted for 8 weeks. Significant increase in p a t h
\\-as observed starting on the 5' week in the treated groups.
b) Rabbitfish
fn.
grow faster in dilute SW than in full-strength SW. .4n esperiment was
conducted to find out whether rearing h) in dilute SW and treating h e m with bovine GH u ~ u l d
have an additive effect on growth. Fry were reared for 8 weeks in dilute SW (20 ppli and botine
GH (0.01 pp'g BBW) was injected u~eeklyin the fim 4 weeks. Rearing the h)-in dilute SW and
treating them also uith bGH did not further improw the growh.
c) Preliminary experiments urre performed to test the effect of he purified GH to promote
grouth in rabbitfish
fn. Weekly
intraperitoneal injections of he GH significantl) a c c e h e d
grouth. The effect uas e\ident 3 weeks after the starl of he treatment. and w a s still significant1
\\eelis afier the treatment u-asterminated (The results are summarized in publication So. I).
-.
Producrion of recombinanr rabbitfih GH
Recombinant rabbirfish GH was cloned in the expression vector PET-3d and used to aansform
BL?lDEI; bacteria. Large amounts of the recombinant protein were produced: he prorein uas
refolded and purified. The refolding and purification steps were monitored by SDS-PAGE and
HPLC. (The results are summarized in publication No. 3).
X Biological acrivir) ofrecombinan/ rabbitfish GH
The biological activity of 2 preparations of recombinant rabbitfish GH (&H1
using rabbittish
fr?-. Groups of rabbitfish Fr?. were
uas tested in r i w
injected with rrfGH-1 (refolded in 0.1 mM
c)-steine) and rrfGH-2 (refolded in 0.2 mM cysteine) at doses of 0.01 (low dose). 0.1 (medium
dose) or 0.5 (high dose) pg'g BW once a week for five weeks. Increases in body aright and body
length wrre monitored during each injection schedule. which is at I-week interval for 5 weeks.
Rabbitfish fn. injected uith the highest dose (0.5 pg/g BBW) of &H
refolded in 0.1 m5i c!-steine
\\ere bigger than h) in all other ~ m u p s The
. size difference w a s apparent 2 \\-eels afier he stan
of the treatment (The results are summarized in publication No. 3).
6
F. Impact. Relevance and Technoloev Transfer
Throughout the project. the research involved a close collaboration beheen he scientists
participating in the project. Recombinant GH was re-folded and purified b!-
he Israeli
collaborators and transferred to the Philippine partner for testing its ability to enhance -gonth in
rabbitfish fi)-. The Philippine Fanner came for a \isit to Israel in order to familiarize himself with
the equipment needed for large-scale production of recombinant proteins as well as to learn new
ways for incorporating the GH in micmdiet of fish larvae. The recombinant rfGH nas used for
enhancement of p w l h of cultured rabbitfish. Dr. Felix Ayson came to Israel in he second >-ear
of the project for training in methods of incorporating recombinant GH polypeptide in micmdiet
of fish larvae as one nay of administrating the hormone. He also n-as p m e n t d to he equipment
needed for production of recombinant proteins so that he mill be able to acquire these items using
pan of the budget allocated for this purpose.
G. Proiect .4ctivities/Oumuts
I . Dr. A>-sonvisited Israel for 2 \veeks in November 2002.
2 . Publications:
Ayson. F.G.. de Jesus E.G.T.. Arnemiya Y.. Moriyama. S.. Hirano. T.. and
Kan;auchi. H. (2000). Iwlation. cDNA cloning. and p n l h promoting activin- of
rabbitfish (Siganus gunatus) gronfi hormone. Gen. Comp. Endocrinol. 117: 251259.
Ayson. F.G.. de Jesus E.G.T.. Moriyama S.. Hoyodo. S. Funkenstein B.. <krrler.
.and
I.
L.
u a u c h i . H. (2002). Differential espression of insulin-like p n t h factor
I and 11 mRNXs during emb~ogenesisand early l m a l development in rabbifish
Siganus gzmarils. Gen. Comp. Endocrinol. 116: 165-174.
Funkenstein. 8.. m-man. A.. Lapidot. Z.. de Jesus-Ayson. E.G.. Gertler. A. and
.4ywn. F.G. (2005). Expression and purification of a biologically a d \ - e
recombinant rabbitfish (Sigonus g~~frrrro~ns)
gronlh hormone. Aquaculture 250: 504515.
H. Project pmductiviIy
\lost of o w goals \\-ere ache\-ed throughout the project although h - o aims were not obtained. In
the original proposal we intended to tv to extend the half-life of the recombinant rabbitfish GH
that \ve produced. This pan was not accomplished for lack of time. Also. in our original plan.^
7
intended to produce r e c o r n b i t rabbitfish IGF-I. Again. due to the short time and limited budgel
provided for this project. we have only cloned h e IGF-I cDNA and characterized in sequence hut
did not produce h e protein.
I. F u m Work
In the laboraton of the Philippine PI. activities toward developing s m e g i e s for incorporating ihe
recombinant protein into formulated feeds for mass application will be continued. Some
important pieces of laboratop equipment necessw for this kind of work ha\-e already been
purchased using the funds provided by USAID. However. outside funding sources will S i l l be
a c t i \ ~ I ysought h>-the Philippine PI in order for him to continue the work we narted using M
from USAID.
s
General ard Cmparauve G d o u u o b 1117.251-259 (i000)
dot 10 I W p 1999 7409. a ~ l l a b l e m l m
at h n p ~ j v v x
tdeal%m
u.l i t tl'
Isolation, cDNA Cloning, and Growth Promoting Activity
of Rabbitfish (Siganus guttatus) Growth H ~ r m o n e ' . ~
Felix G. Ayson,' Evelyn Grace T. de Jesus,' Yutaka Amemiya.t
S h u n s u k e Moriyama.t Tetsuya Hiran0.S and Hiroshi Kawauchit
'Aquarullure Depanmenr. burheart Asian Fisherks kebpmenr Center BEAFDEC AQD). Tmusn 5021 Ibib.
The Philiooinerr
~
d Fkheria Snenm.
~.~ &laem Cnh-w.
~.? Labmatom
- ,of h4olmhr E d m w x h Sdml
Sanriku. {warp 022-0101, Japan: and ?Hawaii lnstiruteofMm.m Bielw, L ' f i ~ ~ d
~ Ht ayw a ~ iC
. m u t Irhnd.
K-,
Hawaii 96744
-
~..
Accepted October 25. 1999
We repon the isolation. cDNA cloning, and gmwth
promoting activity of rabbitfish (Siganmguftahls;Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbidish GH was -ed
horn pituitary glands under
alkaline conditions, fractionated by gel filtration chromatography on Sephadex C-100, and p u d k d by highperformance liquid chromatography. The bactions containing GH were identified by immunobloning with
bonito GH antiserum. Under m d u c i n g conditions.
the molecular weight of rabbidish GH is about 19 kDa as
estimated by SDS-PAGE. The purir~edhormone war
potent in promoting growth in rabbidish fry. Weekly
intraperitoneal injecrions of the hormone signlficanlly
accelerated growth. This was evident 3 weeks aRer the
start of the treabnent. and i t s eRect was still s i m ~ c a n2t
weeks aRer the treatment was terminated. Rabbidish GH
cDNA was cloned to determine its nudeotide sequence.
Excluding the poly (A) tail, rabbidiih GH cDNA is 860
base pairs (bp) long. It contained unbanslated regions of
94 and 175 bp in the 5' and 3' ends. respectively. 11 ha%
an open reading lrame of 588 bp coding f w a signal
peptide of 18 amim acids and a mature protein of 178
amino acid residues. Rabbidish GH ha% 4 cysteine
p a p ha= k n
data w p m * in
&psiled in ihe DDBJ/EMBL/GenBank dalabavr urder Accession
No. AEQ31298.
Aoalentkoendin~flFQ 1-199941457)for thecDNXseoueweo1
rabbithsh G H reponed in h i s paper.
I
The n d m * vq-
.
0
.
vesidues. On the amlna d kvel. nbbidish GH shorn
high Identity (71-74%) with GHs d ocha perifornu
such as tuna. sea laass. ~ U O W
tail, bmim. d 6LpL.
a d l e u (47-49%) identity with sllmonid d carp
Gk. a m - h
Key H b k rabbitfish; growthhonnne:gmth pn*oo
tion
Growth hormone (GH) therapy isarmma\fy used lo
enhance growth in domesric animals and it has also
become increasingly popular in fish in aquacufture.In
hsh. approaches to accelerate grow* by GH adminktration vary from injection and oral administration of
the hormone to uansgenio. Results from several of
these ~riakwere. however. incomiaenL bxpl for
some cases of transgenia. where the fish p m d d
were exuaordinarily large, albeit few (Devlin H d.
1994). intraperitoneal injection of either m ~ r a or
l
recombinant GH yielded s i g n i h t results 4 if the
weekly injections were for more than 5-9 week.
(Kawauchi eta/.. 1986: Sakata et d.. 1993: Guillen U d..
1998). Oral administration of the hormone \vould be
the most practical for mass application in hsh hushandrv
al&ueh the oueslion of* effective deli\-rr.
3. of the hormone wit&,ut it being degraded in the
stomach should be carefully awessed O f o r i y H
~ dl..
~
~
1993: McLean
lgg9).
Slo\v-growjng and economically important fish spe-
cies are good candidates for growth enhancement by
GH therapy. Such is the case for rabbitfish. Siganus
guttatus Perciformes: Siganidae), an important aquaculture species in Southeast Asia and the Arab Gulf
region. A full understanding of the factors that regulate
growth in this species is necessary if we are to design
strategies to accelerate its growth. As a first step to
understanding how growth is regulated in rabbifih.
we purified rabbitfish GH, examined i t . growth pmmoting activity in the fry. and cloned its cDNA.
MATERIALS AND METHODS
Fish
Immature rabbifih (Siganus gunatus: 150-200 g
body wt) were purchased from fishpond owners in the
northern Philippines. Pituitary glands were collected
from heshly killed fish and immediately frozen in
liquid nitrugen and stored at -80'C until use.
Isolation and Purification
Pituitary glands ( I g) were homogenized in 5 ml of
50 mM ammonium acetate buffer (pH 9.0) containing 5
mM phenylmethylsuUonyl fluoride. The homogenates
were stirred for I h at 4°C and centrifuged at 12.000g
for 30 min. The supernatant was applied on a Sephadex GI00 column (1.9 X 100 cm). Proteim wereeluted
with 50 m M ammonium bicarbonate buITer (pH 9.0) at
a flow rate of 15 d / h . Three-milliliter fractions were
collected and the absorbance was measured at 280 nm.
Appropriate fractions were pooled and lyophilued.
and 10 pg of each fraction was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Laemmli. 1970). Resolving and stacking
gel concentrations were IS and 7.5%. respectively. The
gel was stained with 0.25% Coomassie brilliant blue
(R-250) and the protein bands in the gel were visualized after destaining in methanol:acetic acid:water
(2.5:1:6.5).
For immunobloning, protein bands from SDS-PAGE
were t r a n s f e d into nitrocellulose membrane and
incubated overnight at 4'C with bonito GH antiserum
(diluted 2000~).Immunoreaaion war detected using
the ABC kit (Vector Labs. CA).Tn-o immunoreactive
fractions ( G 3 and G 4 ) were further purifd by
reversed-phase high-performance liquid chromatography (HPLC).
Five hundred miaugrams of G 3 and C-4 faclions
war dissolved in 0.1% vinuoroamic acid tTFA) and
war analyzed by HPLC (LC800. Jawo. Tokyo. Japan)
on a TSK gel ODS120T column (0.46 X 25 cm. panicle
size 5 pm) with a linear gradient of 20-8046 acetonicrile
in 0.1% TFA for 60 rnin at a flow rate of 1 ml'min and
aolumn tempramre of l0%. Abmhnm was monitored
at 220 nrn Individual peaks HW collected. lyphhed.
and analyzed by SDS-PAGE and immunobloning.
The N-term& sequence of rabbitfish G H m a s
determined by automatic q-ing
on a S h i i
PSQl gas-phase protein sequencer as described by
Rand-Weaver and Kawauchi (1992).
cDNA Cloning
Total R&A was extracted from I00 mg of frozen
pimitary with Isogen (Nippon Gene. Tokp. Japan)
following the manufacturer's protocol. The concentration of total RKA was estimated by masuing the
absorbance at 260 nm (conversion fanor. I OD = IO pg
RNA/d). First-suand cDRA was re\-tramoibed
from total W A using the F i t S m n d cDSASynthesis
kit (Pharmacia Biotech. Sweden). Rererse namaip
uon was carried out in a 15.~1reaction mixture
containing 2.8 p1 of total RNA (5 pg). 5 pI of bulk
first-wand cDKA reaction mu. I pl of IS m41 dithie
threitol. I pI (5 pg/pl) of Noddm:, b i n a l
primer, and 5.2 pl of DEPC-treated water.
S u degenerate sense primen and two anti-seme
primers were synthesized (Espec Oligo Senice.
Tsukuba. Japan) based on the mast w n w ~ ~ regions
ed
of the amino acid sequence of knoun t e l a GHs to
clone the internal region of rabbitfish GH c D S A
During PCR. the 50-pl reaaion mixture I I pl h t suand cDNA (template). 2 pl each of sense and
anti-sense primers (final conc. 0.4 pMJ. I pI nucleotide
mixture (0.2 mM). 5 pl of IOX PCR buffer (final corn.:
10 m!! Tris HCI. pH 8.8: 50 d l KCI: 1.5 1nk4SlgCI,).
1.25 units of Taq DNA polymerasel war subjected to 35
cycles of amplification: each cycle m i s t e d of I min
and 30 s denaturation (94-C). I min primer annealing
(40'C). and I rnin and 30 s primer extension (72'C!.
Final emension (72-C) was for 7 min. . b o n g the 12
primer combinations rested. the sense primer GH-8
15'-CC(ACG'IlAT(ACn GA(CT)AA(AG) CA(Cn GA3'1 and the anti-sense primer GH-2R 15'-TC(AGTC)
ACVC) TT(AG) TGC AT(AG) TC-3'1 yielded a PCR
product of the expected size after gel electrophoresis.
The rapid amplification of cDNA ends (R4CE)
method of Frohman (1990) using the 5' RACE system
kit. Ver. 2.0 (Gibco BRL, Gaithersburg. MD).was used
to clone the 5' end. Two anti-sense primers; RF-GH-I
(5'-AGA CTC AAC CAG TCG ATA GG-3') and RFGH-2 (5'-GAC AGC AGC TTC ATC ACA GA-3'). were
prepared based on the nucleotide sequence of the
internal region of the rabbitfish GH cDNA. These two
gene-specific primers and an abridged anchor primer
provided in the kit were used for reverse transcription
and PCR. PCR conditions were the same as described
above.
Two sense primers from the 5' end of rabbitfish GH
cDNA were designed. RF-GH-3 (5'-ATC ACA GCT
GAA CTG CAC AA-3') and RF-GH-4 (5'-AGC AAG
GAC TAG GAC CAG AA-3'). and used with Nodd m l s bifunctional primer as the antisense primer in
cloning the FuU-length rabbitfish GH cDNA. Multiple
clones were examined to guard against PCR errors.
PCR produns were eleamphoresed on agamce gels
and visualized by ethidium bromide staining. The
DNA was extracted and purified from agarose gels
using QIAEX II gel extraction kit (Qiagen. Germany).
inserted and Ligated into pT7 Blue T-Vector. and t m formed in NovaBlue competent cells (Fr'ovagen. WI).
The plasmid DNA was then exuacted from the bacterial cells using QIAprep Spin Plasmid Kit (Qiagen).
Nucleotide sequencing was performed using an ABI
Prism DNA Sequencer 373 (Perkin-Elmer. CA).
Biological Activity
The growth promoting activity of purified rabbitfish
GH was tested in the rabbitfish fry. Forty-five fry (3-4 g
BW) were randomly divided into three groups (15
fish/group) and stocked in separate 250-literfiberglass
tanks. Two groups were given weekly intraperitoneal
injections of rabbitfish GH at doses of 0.01 and 0.1
pg/g body wt. The hormone was first dissolved in a
small volume of0.01 N NaOH and diluted with saline
(0.9% NaCI). The third group was injected with saline
solution and served as a contml. Fish in each group
were individually marked by a small cut in the
operculum lo follow growth in each fish. The hormone
injection was given only until the fifth week and the
fish were reared for 1more weeks. Feeding and water
managements were the same in all groups.-~hangesin
fish body weight and fork length were measured every
week. Significant differences in body weight and fork
length between the control and the hormone-maled
groups at each sampling time were determined by r
test after one-way ANOVA (Sokal and Rohlf. 1995).
Percentage data were transformed by arc sine or
square r w t transformations. where appropriate. before the analysis.
RESULTS
kolation and Purification
The elution panern of the alkaline emwrs of rabbitfish pituitary glands on Sephadex Glal column has
already been reported in a separate paper (Ay3on rraaL.
1999). From the elution profile. fraaiom G 3 and C-4
contained protein h d s of molecular weights becum
18 and 22 kDa. and these bands =me immunoreactiw
with bonito GH antiserum. The HPLC profiles of
fractions G-3 and C-4 are shown in Fi. I. \hen
stained with Coomassie's brilliant Mue after SDSPAGE. the peak eluting at 32 rnin contained a band
with molecular weight of about 19 kDa (Fig. W :
rhts
band cmss-reacted with bonito GH antisemm Fig.
2B). No immunoreaction was detected with antisalmon PRL and anti-flounder SL (data m shoam).
The yields of pure GH from G-3and C-4 -ions
\'ere
0.9 and 1.0 mg/g of pituitary
weight). rpspectively.
N-terminal amino acid sequencing of intM pmein
gave no sequence indicating that the amino terminal is
blocked.
cDNA Cloning
A cDNA fragment of 267 bp was amplified in the
first PCR using the degenerate primers. GH-8 and
GH-2R. This fragment corresponds to nucleotides
347613 of the cDSA (Fig. 3). The second PCR using
primers RF-GH-2and 5' RACE abridged anchor primer
yielded a product of 351 bp which spans from the 5'
end and overlaps with the kno\%nsequence of the
OM-
~
P
0
r,
CI
::
5
9
SI
9
~
n
RG. I . HPLC c h w M t q l n m r of haaiom G-3 (A) and C4 (B).
The lracrimn (0 5 mgl were dimlvcd tn 0 I'bTFA k d d a pminm
~
dGH
and somatolarin 6L) is indicavd G 3 lraion was hry subjened to affinity chromatography to removt ihc $>+opmeirs
FIG. 2. SDS-PAGE panern o l rabbihh CH aher staining with Cmmassie's brilliant blue (A) and aha M
antirrmm (B). >I. molecular weight marker OrDa). I . nonteduced: 2. r e d u d uith p-mercaptoethaml.
m
p with
barito CH
GH-S
ffiH-3
-*
+
347
613
+ GH-2R
pajd irrerrrd don:
I
860
t
f i H full I&
.5brMr1!~
dom
FlC. 3. Sdpmaticdiagramshowing he p i t i o n d i h e IhmFCR poducu I p m i a l a d hll-kglhdmerl.TheNkd b m Rprrraurht@on for he m r w peptide and he open box. he signal peptide (sp) The unvlmlatd RT regions in the 5' and 3' ends m h n &
primers d in ~ e r FCR
y
arr also indicated AAP Is 5' RACE abridged anchor primer
569
141
G A T C C U G A C A W I T G I A C G M C T ~ ~ C U ~ G U U T ~ C ~
D P R Q M T E L L A C F R K D M H K V E
FlC. 4. The nudmtide sequence d rabbilhsh C H cDNA and ils deduced amino and seqThe signal pepode 118 Mluo U is
underlined.The amino add residws are numbered Lu'ginning from he m a r w peptide. The IernWdOn mdan i s indjolEd b? an lnstrt T k
Iw qsleine residues and ihe pol.yadenylation signal are in bold.
40
rabbltfish
tuna
Aslan seabass
yellow tail
bonito
tilapia
flounder
chum ralmn I
rainbow trout
carp
NSUUD.2
----- (IT-E----- (IT-E---T-g--Proe-
-----
-----CT-I--N--QL.--OX-LPER
-&T-LPDER
--D--LP-EI
w
rabbitflsh
tuna
Asian seabars
yellow tail
bonito
tilapia
flounder
chum salmn I
rainbau crout
carp
:a0
SCNSDYIRSP
r-..-->
ALIG'GS...
s-s-.--.-.
s-s-.---.
I--
..--..
.......
-......
.
r--..
F------I-F------I-F------I-F------I.P-H- -NFL-F-.--S-V-F----S-V-F----.m-
F-S
3.5
S-S
F-VX'SF...
'
YSL
i-*SL
S-S-N-YS:.
7--...-
rabblcfish
tuna
Aslan seabssr
yellow tail
bonl to
cilapia
flounder
c h m salmn I
rainbox trout
carp
rabbitfish
tuna
Asian seabans
yellow tail
bonito
tilapla
flounder
chum salmon I
rainbad trout
carp
FIG.5.
r)
Alignmcnl of th? amino acid v q u m of rabbilhsh GH W b uhu leleas CHs Caps
ur intiodurrd w
squtnm
alignmnL Dashes (-) indicate amim acid reridus identid lo ihm o l nhbitkh CH.The l o a r i m ofth? b e amino ddtl.F6ar unique lo
r a b b i w GH u boxed.
internal region (nucleotides 1-354) (Fig. 3). To obtain
the full sequence. a third PCR was carried out using
newly designed primers (RF-GH-3 and RF-GH-4) and
Nofldm,, bifunctional primer. Excluding the p l y (A)
tail. the rabbifish GH cDNA consisted of 860 bp. This
was compased of 94 bp in the 5' untranslated region.
54 bp for the signal sequence. 534 bp coding for the
hormone. and 175 bp in the 3' unbarslated region
including the hexanucleotide M T M . a typical pol!adenylation site located 13 nucleotides upstreamof h e
p l y (A) tail (Fig. 4). Thus. an open reading frame of
rabbitfish GH cDh'A encodes a prehormone of 196
amino acid residues with a s i p 1 peptide of 18 amino
acids and a mature protein of 178 amino acid residues.
TABLE
I
Nuclemide and .Amino Acid S e q u m Identity olRabbiu%h CH
Compared w i h Those of Chher Fish Spedes
Sequence identity (%)
Fish species
Tuna ~TiluNlmL h m m )
h i a n seabas (Llsaim+aj
Yellow lail lWda mmownd~~at
~ o n i t o(l(lk&&
Xlapia ((hgcbnmirnilolicur)
Flounder ( P a d d q s dinaur)
Rainbow m r ( ~ a f n m
gaid-ud
Chum salmon (Oncwhww k-)
Carp ( C p h n wpid
Nuclemides
Amino Id&
73.W
73 40
72 88
72.53
70.63
W.27
59.06
58.37
54-57
74.09
72 51
72.51
71.50
69.94
57.51
58.03
58.03
47.15
Nme. Tuna (Sato a d..1988): k i m seaban W b b a d..1991):
y e U m tail Wlahiki rt d..1988): m
u
,(Naro a 11. 1988); lilapia
(Render-Delrueet d..1989): flounder PV~tahikia 11.1989): rainbow
tmut (Agellon and Chen. 1986): chum salmon (Ekine el 11. 1985):
rarp ( K m n ad..1989).
followed by gel liltranon chromatography on Sephadex G-100 and rpHPLC. In each step in the p u r i h .
tion process, rabbitfish GH was monitored by SDSPAGEand immunoblorting with bonito GH antiserum.
The immunoreaction with bonito GH antiserum was
specific: no immunoreaction was dececled with antisalmon PRL and anti-flounder SL. and the intensity of
the immunoreaction was sh-ong. indicating a high
concentration of GH in the e m . This can be exp&ed since GH cells are major constituents of the
pituitary gland. The purified protein was ah confirmed to be GH bared on its molecular weight. Under
nonreducing conditions. rabbitfish GH has a molecular
weight of about 19 kDa. as estimated from SDS-PAGE
This agrees with the mokular weight of M.4 W)a.
From
.-DI\'A
The yield d GH
1.9
mg/g
wl of
gknd ann;at. the yield
PRL was only half that of GH (unpublished resultsl.
We tried to determine the amino acid sequence of the
N-tmnind region d the intaa p u e i n but found it
blaked indicating that the hm anino add ha the
N-lennims is ppglummine This was cmfirmed w*
the deduced amino acid sequence of the GH cDNA.
We have also cloned the rabbitfish GH cDSA. The
prehormone is composed of 1% amino aidr with a
signal peptide of 18 amino acids. The marure hormone
has 178 amino acids. slighrly larger (by 5 amino acids)
than flounder GH, which is the smallest teleow GH
(173 amino acids) known so far (H$tahiki a 11.. 1989).
In rabbitfish GH. there is a deletion of 5 amino acids
(Pro-TvyrGly-hpTv) in position 143-117 immediately preceeding the I 1 amino acid deletion in llounder
GH. This deletion is unique to rabbidish GH since no
such deletion was observed in any known fkh GHs
(Fig. 5). This stretch of amino acid midues is ouuide
the five conserved domains of GHs as denribed by
Watahii ct d.(1989).The biological significance of this
deletion is presently unknown. Single or double amino
acid deletions are ah found in rabbitfish GH: amino
acids 9.103- 104. and 157 (Fig. 5). As in the orher teleost
GHs (Rand-Waver a d..1993). rabbidish GH has four
cysteine midues located in highly conserved positions.
Rabbitfish GH is closer in identity to GHs of other
perciforms like tuna. sea bass. yellow tail. bonito. and
tilapia than to those of salmonid and carp GHs. As is
evident in Fig. 5. except for the highly co-pd
amino acids in 170-180. the sequences of salmonids
,,
>
Sequence Compariwn
The deduced amino acid sequence of rabbitfish GH
was compared with those of other fish species (Fig. 5).
The sequence identity is Listed in Table 1. Rabbitfish
GH has four cysteine residues loeted at highly m
d
positions aa 51.151.168. and 176 (Fig. 4). Rabbitfish GH is
highly identical (>70%) with GHs of more advanced
telensu.suchasseabass.tunayeUowQil.andbonito.and
less identialwith thcseof d m m i d s and carp GHr
Biological Activity
Rabbitfish Fry inpcted with the hormone at a dose of
0.1 pg/g body w? showed sigruficantly higher increase
in body weight and fork length compared to the
control starting on the third week of treatment (Fig. 6).
T
h
e
e inpcledwithO.01 pg/g body w d t h e hormone
showed a siuficantly higher immwaRer the fiRh week
Although the treatment was stopped aRer the fiRh week
the siplicant immw in body weight and fork length in
the hormoneaeated p p s we. mainrairrd until the
seventh week or for 2 weeks aRer the last injection.
DISCUSSION
We have isolated and purified GH from rabbitfish
pituitary glands by extraction under alkaline condition
A
-8-
-E+
25
i
wnuol
f f i H (0.01 pug B Y
f f i H (0.1 pug BW)
f f i H (0.0 1
BW)
-E+ f f i H (0. I pug BW)
5
weeks
FIG. 6. E R e n r o i f a b b i ~CH on growh in body weight (A) and iork length (B)dherabbithrh fry Fish rcrriwd a r o l a l dsix inmp&~oml
injecilornevery 1 d a y . V e n i d lines represent SEhf 'Signifiwntd i l l e r e m IP < 0.05) compared lo ~k
crmrml
and carp GHs are clearly different fmm that of perciforms where rabbitfkh is classified.
The purified rabbitfish GH was effective in promoting growth in rabbitfish hy. Although the hormone
was given only every 7 days. its gmwth promoting
effect was already evident after the third week in the
fry treated with 0.1 pg/g body r~ and starting on the
fifth week in those treated with 0.01 pg/g body wt.
Interestingly. the significant effect of the hormone was
still observed 2 weeks after the final injection. Considering that the hormone was given only once a week.
administering the hormone daily using appropriate
routes such as feeding would probably banslate into
much larger gmu& increments.
ACKNOWLEDGMENTS
HC are grateful to Dr A. T a k M of Kitarato University for his
invaluable suggesionr and technical suppon. ThanLs are alro due to
Roger E
M P. Mamauag and Pauor Jones Denusla of SEAFDEC
AQD for help in pituitary coUenion.
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honnm: Md&
cloning ol cDNA and -ion
in Ewhsirhi3 mLi m A 5.46-77.
A p n . F C.. de Jezur.E C. T.Amemiya. Y. Morlyrma S.. Hinm.
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vrmawlacdn i n rabbi&
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homolqpm
gmwlhhornme.1 Mar Bar&
6.142-151.
ffiwauchi. H.. Ymyama. 5.. Ymda. A. Yamaguchi. K.. Yliraham.
K.. Kubom. J.. and Himno. T (1986). lsolalion and d u r a s i n l i o n
of chwn A w n gmwth hormone. Arch Biochm. B m . 214.
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L. &zm J . H+n& A. md
HCILt J. (1991) M o l r m k r c l m q and
d Amanban
black beam AmrlropqFa burmsiand b a m m d h
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K m . Y. Sarid. S.. Ber. R. and Dank!. V (1989) Cap -rh
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Y)9-3IS.
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assembly d k head d b a u i o p h q g T 4 . h Z Z 7 . 6 S l 4 S 5
Mdran. E. Romhddt. B.. %en C . and NyMuddin kIPP9)~
Canroinles~delireryd~andpaMh~w.qurult
d telemo. A ~ N In.
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Mmyarna. S.. Yamamoco. H. Sugimno. 5. Abc. T.W.T. and
Kawauchi. H. (1993). O d adrmruYRnm d mnnbinn wlmDn
g m w t h h o r m a n e ~ r a i n b m v ~ u ~ ~ & ~ ~
lurr 112 s 1 m .
Noso. T.Yasuda A. Ka-.
1 .-T
H.Tabhashi. .A. W,
K.. and Kamauchi. H. (1988). b h 3 m and durxtarmian d
@mwhhwmone fmm a nwim Tub bonito iKamwmn plunirt.
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Rand-Weavu. M.. and Kauawhi. H. (19923 A Rpid pacdrrr for
Ihe idation of bioaaiw p hhannrr Gsl Lw+ m~
85.34-345.
R a n d - h v u . M.. Kawauchi H. and Om M (1993) E w i m m d
I h e ~ t u r e d i h e g ~ ~ w t h h o r n m e a &The
nd~f~
E n d o c r i ~ d C m u t h D r n b p n a r m d ~ m N r w
bates.' W.P Schreibmam C. C. S a m 6 md
K T P.ng Eds).
pp. 13-42, Aedanic Rar San Drgo.
R a k - D d r u r . F . SuD.. P h i h w J. C. C k . C ~ Lm
.
M.
Benrubl. 0.. and h*mul. J A (1989) T
iw
t
h hmmm:
M d r m k r d w of c0SA md upasa,in Gchf&a d
i
DNAbnl-nB
H~
-la
S.. Nmo. T . M a i y S.
~ Hiruo.
~
T. rd
(1993). noundamhornme.lodmndrm-rd
d e c L s ' m g r o v a h d j u u s l i * r a i n b m ~ a r6
.~
.e
w.
m. M.
Sato. 5.. L 4 b ~ a d ~K.
t M u n u K.
M.
Y.
Kimura S. X w u b M.. and KimA. II-. Y o k c u h c b v l l g
and nwleotide sqequurr drum p hhornme rDNA Bda
A N 909.3542
S e h . 5. b h k a m i T. Nirhi T. Kuwnu. Y_ Luo A. h n U.
Itoh. S.. and Kauawhi H (1985) C h q rd u p a r ~ a nd
cDSA for A w n @mwh hnmm in Errkr*Bu miY ld. .W
A d Sci US4 82. 4)(M310~
Sokd. R. R.. and Rohlf. F J (19951 "Bianmy- 3rd cd. F m m a n
New Y a k
U$W
h4
. . Tanaka. M.. Wzsda X . Y
d m M. Ymrda Y
and Nahhirna. K. (19861 d J S A w a n d pi--umumd
y*llw. la11(Saidr -a
l1
pqmwlh homo"t Gsl Guq
Guiarmd 70. IOI-406
Walahi*. .U.. Y m w m ~ o .M . Yanukana U . Tanalra M. and
Yakarhirna. K 1140I ComaMd and u n t unimr p d rrvdus
1nlhedomuaoIihegmu%~hamMsJ&dChZLI312-316
.General and Comparauw E n d a m l o g y 126. 165- 174 (2002)
do1 10 1006gcen 2002 7788. awlable onllne ar hrrpll-w ~ddnbrar)corn on
1BELL@
Differential Expression of Insulin-like Growth Factor I
and I1 mRNAs during Embryogenesis and Early Larval
Development in Rabbitfish, Siganus guttatus
E v e l y n Grace T. de Jesus.' Shunsuke M0riyama.t
Susumu Hyodo.S Bruria F u n k e n s t e i n . 5 Arieh Gertler.1 and Hiroshi Kawauchif
Felix
G. Ayson.*
'Aquaculrum Depanment. Soulheau kian Fisheries Lkdopmenr Center (SEAFDEC AQDj. Timuan 5021. h k .
Philippine: thborarory of Molecular EndocMolw. .k/xml dFirheries Sciences. I(I~arat0U~JWNN.
hnnlru. Inare
022-0101. J a m !hborato,y dPhjsiorOSy. &an Research ~ m l u t c univcmy
.
ofrohp. %kam. Tokw. Japan.
§Dppanmenr d.Uark B i o l w and Biorechnalogy, brael Oceanographk and Limnobpcal Rsearch.
Tel-Shiknmna. Haifa 3 1080. luael: and lDeparmwn1 oflioc'lemiruy. Fmd Science and Numuon.
Famiry of A ~ M t u r e The
. Hebrew UniveWr). ofjenrralem. Rehovor 76100. hael
Accepted January 17,2002
In rodents. the expression of insulin-like growth factor I1
OGF-11) is higher than that of insulin-like growth factor I
OGF-I) during fetal life while the reverse is true after
birth. We wanted to examine whether this is also true in
fish and whether ICF-I and IGF-Il are differentially regulated duringdifferent stages of embryogenesis and early
larval development in rabbitfish. We first cloned the
cDNAs of rabbitfish IGF-1 and ICF-11 from the liver. R a b
bitfish IGF-I has an open reading frame of 558 bp that
codes for a signal peptide of 44 amino acids (aa). a mature
protein of MI aa. and a single form of E domain of 74 aa.
Rabbitfish IGF-II, on the other hand, has an open reading
frame of 645 bp that codes for a signal peptide of 47 aa. a
mature protein of 70 aa, and an E domain of 98 aa. On the
amino acid level. rabbitfish IGF-I sharer 68% similarity
with IGF-n. We then examined the relative expression of
the two lCFs in unfertilized eggs, during different stages
of embryogenesis. and in early larval stages of rabbitfish
by a semiquantitative reverse transaiption-polymerase
chain reaction. Primen that amplify the mature peptide
region of both lGFs were used and PCR for both peptides
was done simultaneously. with identical PCR conditions
for both. The identity of the PCR products was confirmed
by direct sequencing. Contrary to published reports for
seabream and rainbow trout. IGF-I mRNA was not de-
tected in rabbitfish unfenilized egkr; it wns hexpressed in larvae soon after hatching. ICF-n m m A . hever. was expressed in unfertilized cggs. a b i t w d l y .
and was already strongly expressed during the c l u n g e
stage. m R N k for both peptides upre
expmsed
in the larvae. although IGF-U mRWA expression was
i.
.s-r
*A
higher than IGF-I expression. c ~ cb
Key Words: rabbitfish: IGF 1: IGF-Il; cDNA clcming:
mRVA expression.
INTRODUCTION
Insulin-like grow.th factors I and Il OGF-I and IGFUJare two highly homologous mitogenic peptides that
are expressed ubiquitously and show diverse effects
o n development. growth. a n d meabolism (reviewed
by Rosenfeld and Roberts. 1999; Werner and LeRoith.
2000). The c e n m l role of ICF-I is that it regulates
postnatal growh and mediates the growth-promoting
hormone (GH). although smdies aka
action of g r o ~ t h
show that it is important for normal embvonic
growth (Baker er a/.. 1993; Powel!-Branon u al. 1993).
The physiological function of IGF-U. on the other
hand, is not yet definitely known, although it is considered a fetal growth factor based on the evidence in
mice. where a significant growth retardation was observed in pups born with the IGF-I1 gene disrupted or
knocked-out @echiara et al.. 1990). The biological actions of lGFs are mediated by IGF cype I (IGF-I-R) and
II (IGF-ll/mannase 6-phosphate-R) cell surface receptors. Interaction with IGF-I-R is mainly responsible for
the mitogenic effects of IGF (LeRoith et al., 1995) and
there is enough consensus at present that much of the
biological action of lGFs on growth and dilferentiation
is mediated by this receptor. The role of type 11 IGF
receptor in IGF signal transduction remains controversial (Kiess. 1999: Nissley. 1999). In addition. there is
the insulin receptor (lR)/IGF-I-R hybrid which seems
to be widely expressed and even appears to be the
most abundant form of receptor in certain tissues
(Baiyles et aL. 1997) but the specific differences in
signaling characteristics in the IGF-I-R and the IR/
IGF-I-R hybrids have not yet been established. Furthermore, at least six insulin-like growth factor binding proteins (lGFBPs) are already found in circulation
and they modulate the biological activity of lCFs by
controlling the availability of free lCFs and by prolonging their half-life (Hwa et d.. 1999). These IGFBPs
show contrasting effects on IGF function. IGFBP-I
(Van Buul-Offen et al.. 2000). ICFBP-2 @uan et al..
1999: Hoeflich et al., 1999: Wolf et al.. 2000). and
ICFBP-3 (Hockheid et al., 2000) have inhibitory effects on IGF actions, whereas there is evidence that
IGFBP-5 functions as a growth factor (biiyakoshi et al..
2001).
To accelerate development and improve growth of
rabbitfish (Siganusgunatus), an important aquaculture
species in the Southeast Asian region. we have been
studying the hormones in the pituitary-liver axis of
this fish. We have isolated and cloned rabbitfish GH
and found it potent in accelerating growth in the fry
(Ayson et al.. 2000). In this study. we cloned the
cDNAs for rabbifish IGF-l and IGF-I1 from the liver
and examined the expression of the mRNA of both
lCFs during different stages of embryogenesis and
during early larval development. We report here that
IGF-l mRNA was not detected in unfertilized eggs but
was fist expressed in the larvae scan after hatching.
ICF-11 mRNA, however. was detected in unfertilized
eggs. although very weakly expressed, and was already strongly expressed staning from the cleavage
TABLE
I
Primers Used in Cloning Rabbi&
ICF-I CORA
I. Degenerate primers for doning he lrvcmal rrlpon
IGF-IF 5 ' -GG (CTG) AT
G T C G !TC: GAGTGCTGC- 3.
IGF-ZF 5' CAGAC (CCll CT {CGi TC in,CCCX LC?.-\>-3'
IGF-IR 5' -CC (AC) CA (GA) TACAT ICT! TC LCCn .AC- 3'
11. P r i m for cloning he 5' end'
rllCFI-IR 5' -CAXACTGCACCCTGTCCACC- 3'
rflCFI-2R 5 ' -IUACCCTCTCTCTCCAC.ACAC. 3'
UI. Primm for cloning ihc 3. end'
rllCF1-IF 5' - C A T C T C T A G C G C T C m C C m t - 3'
rflCFI-ZF 5' -CTCATCTCTTCAACAGTCCG 3'
rflGFI J F 5' -ACCAGTGCTCCTTCCAAAGCTG 3'
N. Prime, used for RT-PCR K R pmduct size = 151 bp!
rflGFln-F 5' -TCTCACTACTUTTCTCCATC- 3'
rflGFlfl-R 5' -CTCTTCCCTCGACTTCACTT. 3'
(m
' The abridged a n c k prima pmvikl in he 5 - RACE kmt w a s
used as ihc forward primer
Nod-dm,, b~functionalprimer w a r used as ihc mau prum
'
stage. Similar to those observed in other venebrates.
IGF-U is expressed more than IGF-1 in rabbi& embryos.
MATERIALS AND Ml3HODS
Cloning of Rabbitfish ICF-1 and lGF-U d ) N h
The procedure of Ayson ef al. (2000) in cloning
rabbitfish GH was followed with some modilkations
Briefly, total RNA uas emacted Fmm the liver with
Isogen (Nippon Gene. Tokyo) and h - s p a n d cDN.4
was reverse-transcribed from 5 pg of coral RNA using
a First strand cDXA synthesis kir (.Arnersham Phar.
macia Biotech. Sweden).
Degenerate sense and antisense primers (Tables I
and 2) designed based on the consened sequences of
the B and A domains of known lCFs were qnlhesized
and used to clone the internal regions of rabbifish IGF
cDNAs. A 50+1 PCR mixture using G e d m p ' s .Amplifaq Gold kit (Perkin-Elmer. Palo Alto. CA) icas
subjected to 15-min hot start at 95' and 40 qcles of
amplification: each cycle consisted of I min 30 s denaturation (94"). I min primer annealing (45'). 1 min 30 s
primer extension (72'). and a final extension of 7 min
at 72'.
The 5' rapid amplification of cDNA ends (RACE)
method of Frohman (1990). using a 5' RACE s>Ttem
' ICFs during Early Dwelopmenr In Rabbilfish
TABLE 2
Primers Used in Cloning Rabbidish IGF-I1 cDNA
I. Degemrate primers for cloning the internal region
IGF2-IF 5' -GA (AC) ACGCT (AG) TCTCC (AG) GCA- 3'
IGF2-IR 5' -CCCACAGTACTGTCCA (AG)- 3'
ICF2-2R 5' -ACAACACTCCTC TTC) ACCAT (AC) CC- 3'
11. Primers for cloning the 5' end'
rflCF2-IR 5' -TTCACCTCACAGCTACGGAA- 3'
rflCF2-2R 5' -CTCCTCCTTGGCCT.4CTGAA- 3'
UI. Primers for doning the 3' end'
rflCF2-IF 5' -TCACTACTCCCATCTCACATG 3'
rflCF2-2F 5' -ACTCACmCCCACACCTCCCG 3'
rflCF2-3F 5' -CACAACCGTCCCATTCTACAAC- 3'
N.Primers used f w RT-PCR (PCR pmducl size = 452 bp)
rflGF2R-F 5' -TCACCCTCTACCTTCTGCAA- 3'
rflCF2fl-R 5' -CTGTGCAACATTCCCTCCTC- 3'
'The abridged anchor primer provided in the 5' RACE lut war
used ar the forward pnmer.
Mod-dm,, biiunctional primer was used as the R V e m primer.
'
kit, ver 2.0 (Gibco BRL,Gaithersburg. MD),was used
to clone the 5' end of the cDNAs. Antisense primers
(Tables 1 and 2) designed based on the known nucleotide sequence of the internal region of rabbifih
IGF-I and ICF-11were synthesized. These gene-specific
antisense primers were used together with the
abridged anchor primer provided in the 5' RACE kit
in reverse transcription and PCR. PCR conditions
were the same as described except for the annealing
temperature (50').
Sense primers (Tables 1 and 2) were designed based
on the known nudmtide sequences of the 5' end of
rabbitfish IGF-I and IGF-11. and these were used with
Nod adapter primer as the antisense primer in cloning
the 3' end of IGF cDNAs. Takara's LA Tag kit (Takara.
Japan) was used during PCR. PCR conditions were the
same as described except that the 15-min hot scan
(95') was omitted and the annealing temperature was
raised to 55'.
PCR produrn were electrophoresed in agarose gels
and visualized by ethidium bromide staining. The
DNA was extracted and purified from agarose gels
using QIAEX 11 gel extraction kit (Qiagen. Germany).
inserted and ligated into pT7 Blue T-vector (Novagen.
WI: for partial clones) or TOP0 XL cloning vector
flnvitrogen. CA: for full-length clones). and transformed in JM 109 competent cells (Takara: for partial
clones) or TOP 10 chemically competent cells (Invitrogen; for full-length clones). The plasmid DNA was
then extracted from the bacterial cells using QlAprep
Spin Plasmid kit (Qiagen). lrjudmtide sequencing was
performed using an AB1 Prism 377 DSA Sequencer
Perkin-Elmer).
Expression of IGF-I and IGF-U mRV& during
Embryogenesis and Early Larval Der~lopmentin
Rabbitfish
Unfertilized eggs, embrym at different stages of
stage). gastrula (508
development (cleavage (32-1~11
epiboly), neurula. during tail bud elongation. and embryos with heart beat), and larvae from hatching until
6 days old were collected. Unfertilized eggs meereremoved from the ovary by cannulation I day before the
expected date of spawning. Because of the v q small
size of the eggs and larvae of rabbifih, samples from
each developmental stage were pooled. immediately
put in dry ice. and later stored at -80' until analysis.
Total RNA from the pooled samples (100-300 mg)
was extracted and first-strand cDN.4 was reversetranscribed from a b e d amount of total RNA (5 rg)
During PCR, I pl each of first-strand cD%A was used
as the template in all samples. Primers (Tables I and 2)
that amplify the mature peptide region of rabbitfish
IGF-I and IGF-I1 were synrhesized and used. PCR
conditions were as described except for the annealing
temperature (58') A PCR using first-strand cDNA
from the liver as the template and one without a
template were also included as positive and negatiw
control. respectively. The identity of the PCR produns
was confirmed by direct sequencing.
Rabbitfish pactin was used as the internal standard
during quantification of IGF-I and IGF-U mkVA expression. Using degenerate primers designed based
on the sequences of known teleost pactin. rabbitfish
pactin was first cloned and. from its partial sequence.
sense (5' -GCC-AAC-ACC-CA%-.4.4G-AC- 3')
and antisense (5' -RG-CCA-ATGGTG-ATG-r\CCTG- 3') primers were designed. PCR conditions were
optimized by first checking the Linearity of the PCR
and. based on the results of the linearity check.a PCR
of 40 cycles was used for all samples. PCR products
were loaded into the gel and stained with erhidium
bromide. Quantification of PCR products m-as done
using GeneGenius gel documentation and analysis
system (Syngene. CK) \'slues for IGF-I and IGF-I1
were normalized with that of 8-acu'n.
A U G D C r r r U W r U G ~ W O U ; t T A r U X T - ~
R G F Y F S K P T G Y G P N S R R P R G
---.----------------------!
I - - - - - - - -'-,,-in
..
,
4>5
4;
XAGAZZ~GTCTr~CMAU:TC'17~:~TCC-XWC-;T&77A
TtTT?TiYXTIUGGC&TiA?G4TGRA - r e s t o f 3 ' -Itrai.sla:e2
re;l=r
5: 5
-
8 56
316
-------- . --
FIG. 1. Nuckolide and d e d u d amim acid sequencpi drabbitfuh prrpmlGF-I. The signal peptide (44a)is underlined. the mwurr ~ g a n
(68 a)mnsisung d B . C. A. and D domaim. is in boldfaced kom. and the sir Cys m i d u a d l h e nrature peptide am double undcrlmcd. T k
E domain is composed of 74 a.The amim acids are numbered waning from the malure p p t i d e ~
RESULTS
determined by 5' and 3' RACE. respectively. By connecting- the sequences of the overlapping- clones. the
nuclmtide sequence from the start of the 5' end
through some portion of the 3' unuanslated region of
rabbitfish IGF'I cDNA war obtained. From
sequence. rabbitfish preprolcf-l war found ro consist of
186 amino acids iaa) (558 bp) (Fig. 1). This is composed
of 44 aa for the signal peptide. 68 aa for the mature
peptide comprising B. C. A. and D domains. and i 4 aa
~~
Structurr of Rabbihh IGF-I and
IGF-U cDNAs
The nuclmtide sequence of rabbitfish ICF-1 cDNA
was obtained in three steps. The sequence of the B and
A domains was detemined using degenerate primers
based on the highly consewed region of the peptide.
The unknown sequences in the 5' and 3' regions were
this
ICFs during Early Development m Rabb~lhrh
803
865
-iGAG-~T~TTAT-T5TCT,7WGTUTTTCrCT~
GTCCICTTTGACCTCCCTGC - r e s t of 2' c : r u s l a t e d regicr.
564
FIG. 2. Nudeolide and deduced amim acid wguenm olrabbilfish preprolGF-11. The s i p a l pptide (47 aa) u undaiincd. he r m m Rgm
(70 aa) is in boldlacld leners El. C. A and D domains). and the six CF midues d ihe malure ppide are doubk u r d u i d . The E d o m
is compovd o l 98 aa. The amirm x i & are numbered sraning from thc mature peptide.
for the E domain. compared with the ICFs from 10 fish
species (Fig. 3).
Similarly. we found that rabbitfish preprolCF-I1 is
215 aa long (Fig. 2). The 645-bp open reading frame is
composed of 47 aa for the signal peptide. 70 aa for the
mature peptide (B. C. A, and D domains), and 98 aa
for the E domain. compared with the ICF-11s from six
fish species (Fig. 4).
Expression of1GF-1and IGF-U mRNAs during
Embryogenesis and Early Larval Development in
Rabbitfish
mRNA for IGF-I was first detected in lanae soon
after hatching but not in unfertilized eggs and during
embryogenesis (Fig. 5A). In conoast. IGF-n mRVA
was already detected in unfenilized eggs. albeit with
rabbrtf~sh
glltkead seasream
black seabrear
barranundi
sculpro
f launder
Lllapia
salmcr.
ra:nbcu trout
carp
goldfish
rabbrtfrsh
grlthead seabceam
black se&reaa
bar:zc"tbra
sculp-n
flounder'
trlapra
salacn*
:ark%
troui*
carp
gold€ rsh
-.
- - - - - -- - - - - - - - - - -.
FIG. 3. Alignment of the amim acid sequence of nbbilhsh ICF-I with the lCF-Lr of 10 fch rpecia Caps 0 are mmdxal to n u n m m the
sequence alignment. Dashes (-) indicate amino aid residues identical to Ihose of rabbllfish IGF-I. *:
gmhkd
SFMU w
(Duguay s 11. 1996): black seabream. kmlhoplgna schkgcli (GenBanL A m i o n No. AFUW.73). barnmundi. b olcrilCI 1Xmh.h
Stahlborn a d . 1999):d p i n . Cot- saxpius RoFIbg.Cueni U A. 1998): flounder. Rntichrb~dio~ivmafTawdka a 11- 1998): dhpu Ordmmsb
m s m b i m Reinecke s 11. 1997): salmon ~ y n c h u Ururch
s
( G o U 11.1989): rainbow ~ r o O
um
~ rhp3m
IPltmbbtt a d thcn
1992):carp. Crprinur orpio (Liang s 11.1996);goldhsh. Clnoiw aunm (Kermwnl s 11. 1998). ' Domaim B. C. A and D OmsNuIc I
hMlW
peptide: ' more than one form of E domain is found.
low expression; the expression was already stmng
starting at the cleavage stage and remained so
throughout embryonic development (Figs. 5A and
58). In larvae up to 6 days old. both lCFs were
stmngly expressed, although ICF-I1 is expressed more
than ICF-I (Figs. 6A and 6B).
DISCUSSION
Rabbitfish prolCF-I and prolCF-I1 cDNAs contained
the B. C. A. D. and E domains. typical of ICFs. The
mature peptide of rabbitfish ICF-l and ICF-TJ have the
six conserved Cys residues: Rvo in the B domain and
four in the A domain. The B and A domains of ICF-l
have the highest sequence homology in several fish
species (Fig. 3). Except for a single amino acid substitution in tilapia. the A domain of the 10 fish species in
Fig. 3 has 100% sequence homology. The B domain of
rabbittish ICF-l has a rhreonine that is lacking in other
advanced fish species (Order Perdfo-)
and its C
domain lacks histidine and asparagine. similar to the
C domains of the perciforms. .Amino acid 9 in the C
domain of rabbidrsh ICF-I is proline: this is serine in
all other hsh species.
The mature peptide region of rabbitM ICF-Il (70
aa) is 2 amino acids longer rhan rabbidish IGF-1 (68
aa). After aligning rhe six curnerved Cys residua of
both ICFs, the amino acid sequence homology of rhe
mature peptide region of rabbidish ICF-I and IGF-U is
68%. Like ICF-I. the B and A domains of ICF-U in most
species are highly conserved (Fig. 4) but. in addition.
the D domain of ICF-Il is also highly conserved. The
amino acid sequences of both A and D domains of
ICF-I1 in hsh species known to date haw 100%homolowThe E domain of ICF-l is, however. highly variable
and more than one form can be found in some species:
two in humans (Rotwein. 1986). four in salmonids
u! pauawp l w y s e . ~r h y w 1-331 .uo!le~y![dure j o
00)Jaye paualap IOU 5e.w I! l e w ~ o ha^
l os SEA\
s o ~ q w apue ~ 8 %
paz!lwajun qsy~!qqer u! V ~ ~ U
1 - 3 3 1 ~ 0la.ra1 a q 'Llqeqad
~
-qsaua8ohqwa j o sa8els
waJaU!p aql u! pue q q v q q w j o sBBa p a q w a j u n
u! p a w l a p IOU s e . ~v h w 1-331 ' ~ 8 %pazgwajun
u! pauawp
1-331a J a w 3 6 6 1 'uq3 pm auaaJ3)
~non
.noqlrleJ pue (9661 ' - pIJ uialsuayund weaJqeas
p e w [ @ u! a w u n v y ! q q e r j o a w l w u! pue
. q s a u a W q w a j o sa8ers IuaJaNp 8 ~ n ' p
s a a paz!
-1rslapn u! ldl~suen
1 - 3 3 1 ~ 0masa~d
aql pau!urexa
a.n 'Xpnls luasa~d41UI q n p e a w U! ueql s o h q
-wa a c ( ~u! ram01 Llleraua8 F! U O ~ M U ~ J U O J !SI q8noql
-It!.luawdola.\ap J ~ 0 h q w ap W O U J O j 1lmuodUlj
osle q 1-331 amp .+\w
d d e 11 .(EM[ '.re JJ u o l x e ~ ~
.Ila.\\od :EM[' . ~ I n!
J ?) ~ u a u r d o l a ~ a
pue
p qwo.18 lmaj
p a ~ d w !peq 1-331 8 q ~ e al ! w l e w mnoqs sew I!
lpun qswa%o.hqwa 8 u w p ~ueuodwIOU q 1-331leql
1q8noq1 LlmowaJd mm I! '(6661 "re JJ rnvu asnow
u! ~ u a u ~ d o l wpue
p qw0.18 l e u o u JOJ Iuaplyns
F! s a w ~pedaqucww a y 1-331 1mp pamoqs &rus
IUe @ n q p 'uaw w ~ W T P
u!~1-331
J >pedal(
uo ~3 j o a~uapuadapaq, j o ammag 'qsy u! osle l n q
slewwew u! Lluo IOU
Kuew u! pauuyuo3 uaaq
a w %u!le!paw U!
Jseq ~3 j o uaya ~U!IO~OJ~-~IMOJS
pue q w o B jeletumd 8upelnBaJ u! 1-331j o aloJ a u
-ap~daa
dnleu
a w w o ~ paAeap
j
s! I! Jalp r ( l ~ p ~
le~@olo!q
e
ssmd
01 I! ~ oJOj t g w o p 3 a w ir! s u o j aldglnw j o w o d a ~
ou aJe aJaw .JaAamoq '11-331 JOJ xwaaks sno%lo
- m a q u! uaAa r(lmp~eJ N ~ ~ O I ! ~m o d u p m o p 3 j o
s ~ u ~aoaj ~ ws.lnon w o q w j o sappdad ~ u e u ! q w o m
I ~
p a uIo d a ~(6661) .re JJ ue!L pue 'palep!we uaqm
~!ua8ol!ws! 91-331u m n q o fflewop 3 a w j o appdad
~ p a w u K se l e w umoqs aAeq (1661) 'p JJ p a u j 8 a t ~
'r(lmp~ep~@olo!qawos ssassod osle L a u .(g661 "re JJ
LenBna) 8u!ploj IJ~JJOJ 10'8u!ssa>o~d'uodnren Jelnl
-laJesu! J O ~~ u e u o d w !aq Lew u!ewop 3 aql .appdad
a w j o ~ s atp
a ~w 0 4 a8meap !SI 01 ~ o u dimp pz!s
- a w d q s! 11 .sanp!saJ ee p ~seq s q pue qsyl!qqel
u! punoj q u!ewop 3 j o u o j auo ,41110 '(8661 " p
JJ q e u q :6661 'PJJ w o q l v l s ilnl(ulX) p q pue z q
uourles 01 8u!puodsauo~ JapunoU pue Ipunureueq
ir! o w Pue ' ( ~ 6 6 1' U ! I A ~ PW ~!I@M
:6861 '.pJJ oe3)
.apodad un~m
a(, amlmum nnewop a pcre 'v '3 ' 8 , -(z&I 'uaq3 pue
5 m n v :(SZZL~X
-oh: u o F w a v i(uesu3) e u l W
n
w
g .uourles w n v :(MI 'PP " ~ ~ ~ - ~ u w o - I )
1rqq-61
mdma -3
u d m -us1'Pr w3)"Qs?m.pcw . q d e n ~:Us61 '.P P ~119)
m j w l o S J J '!prmurerreq
~
:(9661'-FP
ken%)
01
pnuap!
s
n
p
!
s
l
p!x
w
!
w
a
l
w
w
(-)
a
q
s
w
1
v
a
d
!
p
amanbx
np~yllls
:nmqI I - ~
W!WU
~I p
-arwdzynl ns JO ~ I I Jw
~qllm
I n-rl31w ! q q = JO axanbas PW w.wa(,JO IU~&!IV -b
atp anumvl m panparrn
u
~~
:no;a n c q 3 r e 1
u q e s mq3
--q-m-------------------------------------------~-3--d-q-q---~-----------.
u1a:n3s
.-------~------~-yi-y-------------------------------~-~~--~-~---------------e r d e j?a
-----.---------------------------------------------O--d-q-l----.----------:pKnlrrr=eq
--------------.---i----..----------------------.---~----4-~d----l---------w a r w a s peag2:yb
~ W ~ ~ ~ ~ ~ : ) I T I C I ~ . W W ' . I Y ~ S W ~ W ~ X ~ W S U ~ ~ ~ S A ~ ~ XY s~? iYl ? ~
q qW
Pl ~ I A ~ S
----~--------A------.;-------------------y----------.--d+~-~~-~--I---------
----~--------.~------~-------------------~----------.--d-ao-~i-~~-~---------
.
..
II
ILI
qsy~!qqqu! luaudo~a~aa
4lqj h m p 1-31
A IGF-I
FIG. 5. ICF-I and ICF-U mRNA expression during rabbi*
embygenesis determined by semiquanlilalive KT-PCR. (A) Agamse
gel elecuuporesis of RT-PCR produrn lor ICF-U and Baclin. No
PCR product for ICF-I: m. marker 4.
u n f e n i l i eggs ( v e q faint
band la IGF-In; c. cleavage sage (32ceII wage): & g m k : n.
neurula: [be. tail bud elogation: hb. embryo wilh heart k t : nh.
newly hatched larvae. El) ICF-I1mRNA levek exprezvd a the ralio
of ICF-U and Banin. Labels are similar to chat in A.
rabbitfish Larvae soon after hatching. In their studies.
Funkenstein et al. (1996) and Greene and Chen (19971
used a higher concentration of total RNA during the
reverse transcription step--20 pg. which is fourfold
higher than that used in the present study.
On the other hand. IGF-I1 has been postulated as a
fetal growth factor. Support for this hypothesis came
when targeted disruption of the IGF-11 gene resulted
in fetal growth retardation-mice lacking the IGF-U
gene were 40% smaller at b i i than their normal
counterparts Oechiara et al.. 1990). The reduction in
the growth rate in mice lacking the IGF-I1 gene is
confined only in utero, clearly demonstraring the dependence of embryonic development on IGF-11. In
addition. postnatal overexpression of IGF-II does not
result in increased somatic growth, whereas mice
overexpressing IGF-I are larger than the normal type
and showed signs of overgrowth after 3-4 weeks of
postnatal life Wanhews et al.. 1988). These findings
and the generally higher levels of IGF-Il in the embryos compared to adulrs in many species argue for irs
importance in early lie.
The observation that IGF-Il expression is higher
than IGF-I expression during early development
seems to be true also in fish. as shoum by our present
resulrs. IGF-I1 mRNA was strongly expressed in rabbitfish embryos during the cleavage stage. whereas
IGF-l mRhlA was first detected only in the lawae soon
after hatching. Furthermore. IGF-II mRXA was also
detected in rabbitfish unfedized eggs. albeit weakly.
whereas ICF-I was nor. Different obsenations were
made by Greene and Chen (1997.1999) who found not
only IGF-11. but also IGF-I. mRNA in rainbow trout
unfertilized eggs. Nevenheless. the absence of PCR
products for IGF-I in unfertilized eggs and during
embryogenesis in rabbitfish does not imply pmr quality of RiVA from these samples or low efficiency in
reverse transcription because a PCR product for rabbitfish pactin was amplified in all samples and there
was no apparent difference in their expression in all
stages. In the larvae. although ICF-I1 mRUA Iebvls
R u i n
1ISb-
FIG. 6. ICF-I and ICF-II mRWA uprrnm during ca+ Land
development in rabbilfuh d n e n n i d by mqwutatn~
RTKR
(A1 A ~ a m w
gel elecvopmis 01 KT-PCR poducu lor ICF.1. ICF-U.
and baain; nc. ncgatlr? conlrol: liver. ~ I U M
& IB &F-I
and ICF-I1 mRYA lewk expressed as ihc raD u1lh Bxtin
were also higher than those of ICF-I during the first 6
days after hatching. the reverse is true in the adult, as
shown in the higher level of ICF-I mRNA in the liver.
Overall. the findings of the present study seem to
agree with the concepts that ICF-II is probably more
important than ICF-I during embryogenesis and that
IGF-I exerts more influence on growth and development in later life stages.
ACKNOWLEDGMENTS
We lhanlr Dr. A. Takahashi of Kitasato University and Dr. Y.
Takei of Orran Research Institute. University d Tokyo for heir
helpful advice and s u g g m n s . F.G.A. is grateful to the Japan
Society for the Promotion of Science for the powdatoral fellowship
and for the financial suppon in the conduct ofthe wudy. Thu work
was supponed in pan by USAID (Grant No. TA-MOU-99C19-001.
Program in U S - h l Cooperative Development Research h
gram. Emmmic Gmwrh). We also lhanlr his. F. P e d m and E. M.
w a g of SEAFDEC AQD for their arskunce.
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functiorn as a growth factor. I
.
Cljn Inw. 107. 72-81
Nissley. S. P. (1999). Type 2 ICF receptor-mediated evenn. I n -The
ICF System: Molecular Biology. Physiology and Clinical Appl~catiom* IT. C. Rosenfeld and C. T. Robens. Jr.. Eds.).pp. 165197.
Hurnana Pren. Tolowa NJ.
Powell-Branon. L. Hollinghead. P. Warbunon. C.. Dowd. hi..
Pitts-hleek. 5.. Dalton. D.. Cillett. N.. and Stewan. T. A. (1993).
ICF-I is q u i r e d f w normal embryonic gmwh in mice. Gnu
LLv. 7.2509-2617.
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nrssambin~the lilapia: Gcne sequena. t i m K w o n . and
cellular bcali+alion. Endmumlogy 138. 3612-3619.
R d e l d . R. C.. and R o h C. T.. Jr. (Eds.1 (1999). -The ICF
Syscem: Molecular Bidogy. Physiology and Clinical Applicatiom.' Hurnana Ren. Totowa. NJ.
Rotwein. P. (19861. Two m l i m l i k e gmuzh f x ~ u
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RV.& are exin human L b u . R u i ? l A d Sa LS.\ 8 3
77-81.
Shamblolt M. J.. and Chen. T. T. (19921. !dmtITwMm d a m d
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Siegfried. J~ XI.. Kaspnyk. P C.. T r m o n A. Y . Mubhur. I L.
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f m o r IB pmhomarr. Pm .%d~ A d So.L-$4 8%3107-8111
Tanaka. M.. Taniguchi. T.. Yamarnolo. L. Sxkagm!!. K ~ Yoshuato.
.
H.. 0hkubo.T.. and N
K (1998). Gcne and DZIA wruc.
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T. T. (1999). Recmnbirwt E-pepidcr d p l C F - I h a w m t o g a u
aaiviy.
110. 3387-3X.
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C . h i . . B l a m e n R J . . K o e d a m J A . . a n d \ ' m W J . W 11(ml
Human insulin-Like gmu* factor UCFI hndirg pmarr I h b ~ u
ICF-I rtimulaled body growlh but somukvs ~ I I dI the Mney i n v ~ ld lu a r f m m
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rpbcmg p a t h n a ? ~ ~
.Ud. E n h r i m L 7. 409-422.
Werner. H.. and LeRoith D. (m00) .New 0 0 1 1 ~ i
~n(mguhnm
~
and
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932-942.
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Yakr.S..Liu.J.L..~B..&I~.A..AmhD.Lus.B.d
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Available online at ww*vxiencedirect.com
Expression and purification of a biologically active recombinant
rabbitfish (Siganus guttatus) growth hormone
Bruria Funkensteina-*,Anna Dymana, Ziva Lapidota, Evelyn Grace de ~esus-&son~.
.Geh Gertlerc, Felix G. son^
and E ~ o t c c h n o l .Vanonal
~:
lmnrvv 4?
-.
l r d
ad
T e I - % h a . PO &u &l30. Ho./a 3la60. l r w l
' l q u u m l m L k p m m c S o u h r .am Fishme brl0p"m Cenm 6E4FDECIQDl. 7igbrmul .W2l l h i b
'lnnrruw o j E ; a h m u n ) : F o d S ~ ? F . ~
18nd
I HUmlm. Fa+
0J.AFood d fiwoowwmd
a
The Hebrp* L'nr,m#nqTJemalem R c b w - 6 l M l r d
'Lkprrnnenr o j . L h i n e E i o l w
Rccrlred 6 A*
2001; rrcclred m r e d form I ? .-I
?LW.
R m a
n
amprsd 20 Apnl ? W 5
+
Rmmblnanl rabbi& p t h homune(&if)pvlein was
in EicherdP col;. B L ' I (Dm)
alk Tbe CDXA a u o d m p
rhc marure protein ofrRjH was fim cloned in pGEhl-Teasy vector and then t r a n s f e d w PET-3daprssim %amEqmsmn m
E~coli cells was then induced by IPTG (0.4 d l ) . Inclusion bodies (18) containing thc expressed polein nut pnhl* n u M g
bacterial cells pellet with l)mzyme followed by repealed washings in cold upler containing T r i m S-100. sauarru~lad
cenmhgalion. 10 were tben mlubilued in 4.5 h l uru, mfolded a1 pH 11.3 in thcpaena o f a n l y t u mounts o-.f
lai
purified by @Sepharoscmlumn. Gel filtrationon Superdex column shou-edtbe pmfied w
n
i w be a man- GH. M on
SDS-P.4GE, the puriry ofthe recombinant rRjH prrparadon i s appmumawly Ba;. The rocanbinanl f f i H n u tcncd k w IB
btological aniviry both i n vim. by its ability u, nimulaw IGF-I mRNA cxpazicm in tbc liva. lad in \i\o. by lo cb~llc) I@
accelerale g m w h in rabbifish fry injccled with the hormone. A sign~fican~
inm gmwlh was bedm nbbilfid fr? pbm
the recombinan1 h o m n e . P o l y c l o d antibody raised against the native f f i H immumrurod -4th dx r o ~ l b iffiH
~ l ha
bloa and in ELISA. indicating thc suibbiliy o f thcw reagents for fuiurc q w t i h o n of C H in rabbiIfifish p
h
t 0 0 5 Elwvier B.V All righb r m d .
-' Concspad~ng a u h Tcl: 4 7 2 4 8565233. fax: -72
8211911~
E-moil d h : [email protected]~[B F~mkcnsldn).
Gmuth hormone (GH) is a singlcchain polgepti&
4
of about tt kDa prodwed and w e d by sonwcmphs in the antenor pad o f the pituitary gland. GH
plays an essential mle i n stimula~ingromalic _mum in
vertebrates. Numerous studies have demonstrated that
injection of narive or recombinant GH to different fish
species resulted in -mwh enhancement (runkclljrzin.
2'H10), indicating thar increased levels of GH have the
potenrial to improve ,guuzh in fish.
The rabbitfishes (Perciformes. Siganidae) have long
been considered a good candidate species for mariculNre because of their capacity to breed in captivity
consume a wide variety ofalgae in the natural environment. can be readily weaned to accept formulated feeds
in confinement, and high tolerance to environnlental
conditions (L3111, 107-1). The 26 species of rabbitfishes
found in the Indo-Pacific area (\lwud;l cr 31.. I'J8-i) are
all excellent food fish and among them, the S~ganur
g~~rrarus
is best suited for mariculhlre because of their
large size. Consequently. lhis species has been the
subject of many research studies in SEAFDEC AQD
resulting in the development of technologies for c a p
tive breeding and hatchery production (Du?. I ' M ) .
We have been trying to accelerate grouzh in S.
gurrarus in order to reduce the culhlre durat~onto
reach marketable size. Towards this end. we med to
identify the limiting component(s) in the g r o u ~ haxis
(GH-IGF-I axis) in this species in order to be able to
recommend to the fish farmers some interventions to
make this species prow faster. .As a first step. we have
cloned and charac~erizedthe GH family of proteins
(GHprolactimsomatolactin) and insulin-like grouzh
factors (lGFs) in S gvrrorur (-4yson ct 21.. IqW.
:In.)l1. :111)3). This allowed us to look at gene expression panems of these hormones during various developmental stages. In those smdies, we found that
mulriple injections of purified native rabbitfish GH
(&H) ro rabbitfish fry stimulated u-eight gain, indicat~ngthe porential of GH therapy to enhance prouzh
in th~sfish species. However, obtaining even a small
amounr of narive GH requires the sacrifice of enormous mlmben of fish. In lhis paper, we Rpon on
cloning. expression and purification of a biologically
active recombinant rKjH in E coli cells.
2. Materials and methods
Oligonucleorides were prepared by Sigma (Israel).
Resmction and mod~kingenzymes were purchased
from Xew England Biolabs (Beverly. %fan)
and Promega (Madison. W. All other chemicals were o i
analytical p d e .
2.2. Construction of rJ@H erpression wctor
A fragment of rfGH cDNA codins for manue
GH protein was amplified usxng rfGH cDS.4
(A~solrer al.. X W t ) as the templae and the pnmcrs
shown in Fie. I . The upmeam PCR prima cornsponded to amino acids I 4 of &H and COnlauKd
an additional alanine and the .Vml resnicrion site.
which donated the ATG codon. in the same ding
frame as the rfGH. The downmeam PCR p
k
corresponded to amino acids 173-178 and included
a stop codon immediately aAer the last mdon. iollowed by a BamHl restriction site. PCR amplificb
lion was conducted in 50 111 using 2.5 L' Taq
Polymerase (Sigma. luael) and 30 cycles. as fob
lows: I min at 94 'C. 1.30 min at 56 'C. 2 min at
72 'C except for the first cycle for 3 min af 94 'C
and the last cycle of 10 min at 72 'C. Thc amplihal
fragment was purified by gel elecoophorsis. followed by elunon using QLAquick Gel Exnacbon
Kir (Hilden. Germany) and cloned in pGE.\i-Te-vecror (Pmmega Madison, W.1)using JM109 E cob
as the hog bacteria. R c ~ a
of an LllM w m
verified by digestion uith EcoRI rcmiction -me.
One positive clone was seqmced with SP6 and r
RKA pmmoter primas to verify IIW
m smrs
occumd during PCR amplification and cbning.
The insen of the malure GH vas cut ow fmn dm
plasmid using .%I
and h H 1 &aim q m e s
and cloned in the expression vector PET-3d d i g c s d
also with >VcoIand B m H l and used to tnrrsfmncd
&I109 E coli bacteria For expression, the plasmid
PET-3d containing rfGH from three clones uas solated and used to transform the hoa bactena
B U I p E 3 ) E coll. b l a b x i colonies that e x p r e s d
the protein after indwnon by IPTG were s e w for
purification. The p m c e of rfGH p t e i n in the
colonies uas checked by SDS-PAGE and \Vestem
blotting using homologous anrivnun (uc below).
The DNA plasmid of rfGH-PET-3d clone that g x e
h e best expression uas sequenced using F RXA
promoter primer to confirm drar the frdgnmt of
f f i H disated with Sdel BamHI mas cloned axrrclIy inro PET-3d digested uith Sdel BamHI.
Rabbitfish G H cDSA
-
.YCDl
5 CGCGCC ATG GCG CAG CCA ATG ACG GAC ACT- 3
Ma Ala Gln Pro Ma Thr
Sn
1
2
3
4
6
&.-H I
3 ETT CCC TTG ACG TGG AAC ATC C ~ K G C G 5
Glu 4h Asn C ~ JThr Lcu slop
1-3
174 1-5
1-6
17-
178
2.3. Erpression. re/olding and purification o/ rfGH
ln a preliminary experiment, the lhree recombinant
bacleria were g o w n in 5 m l L B at 37 'C lo an OD,
of
0.8 and then IF'TG was added to a final concenmtion o f
0.4 mM. Bac~criawere p u n Tor an additional 3 h.
centrifuged for 5 min at 11,000 rpm in a chilled microcentrifuge and h e pellet was resuspended in 500 p l TE,
pH 8, washed twice in the same solution, sonic&
twice for 30 son ice, cenmfuged again and the resullanl
pellet of inclusion bodies was resuspendmi in 100 o f
Laemmli's sample buffer containing SDS and &mercaptoethanol. Samples were heated at 100 'C for 5 min.
chilled on ice and then cenmfuged briefly. .An aliquot
o f I 0 p l was analyzed by SDSPAGE, on a gadienl gel
5% lo 15% One gel ua stained with Coomasie
Brilliant Blue R-250 (Sigma, Israel) and the second
was subjected lo a U'eslem blot using homologousantif f i H antibody (see below) raised againsl native f f i H
(\!.,!n
s[ 31.. 20()0). Two clones expressed f i H (-2 I
kDa). which reacted with the antibody. Clone =2 u-as
chosen for large-scale prepantion.
0
pGEM-T(3018 bps
I
For large-uale prcparation. B G I ( D E 3 I E d i
clone 4 was gmun in 20 x 500 ml LB in dw pmsmx~
o f ampicillin (100 pgtiml) at 37 'C in 2-L flasks lo anof
ODm 0.8. Then U'TG u-as added lo 0.4 mu.The a l l s
were p u n for an additional 4 h. p e l l e d for 5 mm a
10.000xg in a Sowall RC5C cenmfuge at 4 'C and
s t o d at - 20 'C. Fmzen pellets o b i n e d from 10-L
culture (-30 g) u-ere hawed and &bided into L allEach pellet uas f i t s 1 musquou. in w o I-L Ma.
pended thoroughly i n -20 ml m l d distilled uatcr. hen
380 ml o f 10 mW EDT.4. 10 mhf Tris-HCI. pH 8.0
were added while mixing conunwusly with a glass rod.
Lysoqme (0.5 mg'ml) was dissolved in asmall amount
of HzO (200 me400 m l o f baclerial soluoon) and
added. The soluuon uas ~ncubavdfor 30 min at 4 'C
with continuous nirring. The e l l s were thcn sonicaud
for 6 min wilh onoffpulscs of0.5 s and pelleted f o r j 0
min. 25.000xg. at 4 'C. The inclusion bodies ilBI
pellet conraining h e f i H was rrsuspended in cold
dislilled water containing 0.1?& Triton X-100 and sonica~ed.pelleled and resuspended again in Tnlon S-100
containing cold uater. This pmedure u-a-led
several times, until a clean white pellet was obtained.
Last two washes were carried out without Triton. The
final 1B pellet was rauspended in 100 ml water and
divided into 20 m l batches and kept at -20 'C.
I n order to optimize refolding conditions, the IB
pellet obtained From 2 L fermentation culture (20 ml)
was divided into 4 aliquots. Each o f the aliquon was
solubilized in 4.5 M urea buffered with 40 rmU Trisma
base ina total volume o f 14 ml. The pH was adjusted to
11.3 with 2-3 drops o f 2 N NaOH, resulting in cleanng
the solution and then cysteine was added to 0.1 mu,
0.2 mM, I mM and 5 mM. The solution was stirred at 4
'C for I h, diluted with 2 volumes o f cold water and
dialyed for 48 h against 5 x ? L o f 10 mhl Tris, pH 9
and applied to a Q-Sepharose (fast flow) (Pharmacia
LKB Biotechnology AB, Uppsala Sweden) column
0.8 x 4 cm, pre-quilibrated with the same buffer at 4
'C. Elution was carried out using a&wontinuous NaCl
gradient in the same buffer and I-ml fractions were
collected. I n thae experiments most ofthe &H eluted
with 100 m.U NaCI. I n subsequent refolding experiments the protein was refolded in the presence ofO.2
mU cysteine but dial@ against 10 mU Tris, pH 8.
The elution was carried out by discontinuous KaCI
gradlenr and the f f i H was eluted with 50 mll NaCI.
Protein concentration was monitored at 280 nm and
monomer content was determined by gel filmtion
chromatography on a Superdex 75 column ( I x 30
cm) (Pharmacia L K B Biotechnology .a)
In.
addition,
aliquots ofselected fractions were analyzed by SDSPAGE (see below).
2.4. SDSI14GE and lYesrern bloning
The presence. purily and the amount o f protein
recovered from the IB and during the puritiwtion
steps were monitored by SDS-PAGE using 5-15?6
gradient gels. Samples were mked in Laemmli sample
buffer with or without i5-merCapt0~hol. Gels were
stained with Coommie Brilliant Blue R-250. For
Westem blot analysis. proteins were elecno-transferred
to nitrocellulose membranes (Schleicher and Schuell,
Dassel. Germany), blocked in 0.5% BSA in TBS (100
mX1Tns. O.%NaCI, pH 7.5) and immunoreactedwith
a 1 :5000 dilution ofspecific polyclonal antibody nised
in rabbits (see below) against native purified &H
\
I 1 I ! . fulll).Membranes were then incubated
with biotinylated goat anti-rabbit $,I
(H- L). affinity
punfied (Vector Labs. Burlmgame. C.4) &luted to a
final concenuatlon o f 0.15 p g ml. k e c n o n was x compl~shedby the akidln4lonn reapent Weemlam.
.XBC k~t.Vector Labs) and >,Ydlam~nobemdlne In
H202
Antiserum against &H was r a i d in rabbits. ?;alive rfGH (100 pgml) purified from the p i l u i ~
gland (;\yx,n el 21.. 3Mh')) was dissolved in d i e
solution and was emulsified in an qua1 volume o f
Freund's complete adjuk-ant. One milliliter o f the
emulsion containing 50 % o f the antigen was injmed
inuadermally into the exposed skin at the back ofthe
nbbi~
Booster injections o f the antigen (50 ug m l o f
emulsion in Freund's incompleie adjuvant) m-ere then
given inbadennally into the b s k o f the rabb~nfor
four timer at intervals o f 3 week. .4 mu& afier the
last booster injectioh blood was colkcted from the
heart Serum was sepameb lyophilued and stored u
- 20 'C until use. Two rabbits w a e used to pcneraa
the antiserum.
Wells ofELIS.4 plates a!-shapedm M u r %Z?iC.
Denmark) were coated with 0.1 m l o f raanblnant
f f i H (20. 10. 5, 1, 0.1 and 00.01 llg'ml in 0.1 M
castmnate buffer, pH 9.8). incubated o k m i g h l at 4
'C. bed with 1 0 . formaldehyde for 10 mln u 4 'C.
and then washed with PBS containing O.OS?%Twsm 20
(PBS-T) four t i m a for 5 min och. Wells wae p a coated with 0.15 m l o f 49.0 bovlne serum albumtn
(BS.4) in PBS-T. Folloming overnight incubation at 4
'C and washing. 0.1 ml o f rabbit anti-native punlied
f f i H was added ( I : 1000. 1:5000. 1: 10.000, 1 :20.000.
I:40.000, 1 :80.000. I:100.000 dilutions in PBS-TI and
incubated for I h at 37 ' C Afier mashing. goat ant!rabbit IgG conjugated toalkaline phosphamse(Car KO.
IIS-055-144. Jackson ImrnunoR-h
Laboratories.
Inc. W a t Gmve. PA) was added to each well and
incubated for I h at 37 T. Following washing. 0 ~ 1
ml o f subsmte for alkaline phosphawu. p-nitrophenylphasphates (pNPP; Cat. No. 104-105. Sigma) mas
added at I mg'ml to each wrII and incat 3' 'C.
Contml wells did not contain rtGH or no anti--.
The
optical densit). (OD) was determicd using a micro-
ELIS.4 reader at 405 nm. Each ELlSA result was
expressed as the ratio between B e mean absorbance
o f tested sample in positive (P) and the mean absorbance in negative (h?wells.
2.7. Biological acnviy
Two batches o f f f i H preparations were used sep
arately for the i n vivo and in v i m bioassaj-s.
2 . 1 1 . In wrm
The procedure o f \loriyalla er 31. I1lXl21 was followed with some modifications. Rabbitfish fr). u i t h
body weight ranging from 13 lo 15 g were dissected
and the livers removed. The livers were washed twice
u i t h sterile water and three times with the sterile medium before cuning into pieces (roughly 0.5 cm size)
and incubated in v ~ t r oin Medium 199 (with Hanks'
salts and L-glutamine, without sodium bicarbonate)
with or without recombinant f f i H . The medium (pH
7.4) was enriched u i t h oxygen and supplemented with
I00 U'ml penicillin. Liver slices were distributed individually in 24-well culture dish containing I ml ofthe
medium. They were pre-incubated for 24 h in the
medium without the hormone. Incubation was catried
out at 18 "C with slow shaking. The medium u;rr
changed the following day with the same medium
containing recombinant f f i H at the following concentrations: 0 (control). I.10. 100 and 500 ng!ml. Incubation was continued for another 24 h after which the liver
slices were immediately put into RNAlateF solution
(Ambion, USA) and stored a1 -20 'C for RT-F'CR.
Liver slices h m three individual fishes were used for
each concenhalion o f the hormone tested.
Increased expression o f IGF-I mRY.4 h m the liver
slices incubated with the medium containing recombinant G H war d e n as a measure for the bioactiuir). o f
the recombinant GH preparation. Tow1 RXA from the
liver was extracled using T R l z o l ~Reagent (lnviuogen, Carlsbad, CA) and first strand cDNAu.as reversed
uanscribed !?om equal amounts o f total RKA (5 pg)
using a cDKA synthesis kit ( h e r s h a m P h a c i a ,
Sweden). PCR was performed using an aliquot o f I
*I o f frst strand cDWA as template and the following
primers (forward: 5'-TCTCACT.4CTGCTGTGCATC3'; reverse; j%TGTTGCCTCGAClTGhGTT-3) specific for B G F - l (.X><nner 31.. ?l)i12). .4 PCR product o f
454 bp i s expected using these primer pain. Th~ny-tive
cycles o f F'CR were performed u - ~ t hthc fullouing
conditions: hot stan 9J 'C 30 s. denamation 9J 'C
30 s, annealing 58 'C 60 s, exunsion 72 'C ik) s. final
extension 72 'C 7 min. The idmtiry o f !he PCR prcducts was confirmed by d i m ! sequencins.
Rabbitfish pactin uzs used as the Internal d a r d
during quantification. Primasspecific for rabbltfish $actin u-ere used: s e w (S%CCA4C.4G<jCl.l.14AGATGAC-3) and anti(5'-TTGCC.AATGGTGATGACCTG-3) (A:-son e l ~ i . . 001). F'CR pFJducts
were loaded onto 1% agarox gel and named u ~ t h
ethidium bromide. Quantification o f PCR +-ls
was done using GeneGenius gel documen~ationand
analysis system (Syngene. fK).Valf w IGF-I uar
normalized with rhal o f pactin.
2.72. In v i w
A total o f 120 tabbitfish fr). u i t h body wright
ranging h m 3 lo 5 g u-ere randomly divided Into
four groups with 30 fry per group. The fn- in m h
group were stocked i n thrrc vparate libaplass mnks
of25O-L capacily or an equivalent o f I 0 tiy per lank. A
total o f I 2 tanks were used for the four groqxs A11 the
tanks were filled u.ith manual seawiuer and u w e held
indoors. Three groups were given intqmiloneal iqection once a week for 4 uacks o f recombinant f f i H a
dosesofO.01.0.IOand0.5~gBW.Thehormoneu~
dissolved in saline solution (0.9% SaCI). The fourth
group (conml) was injected u i t h an equal volume o f
saline solution. The fry were fed tabbiduh anirutal diet
at ja%
BBW per day. The feeds were given at duct rations
at 9:00 AM, 11:00 AH and 2 9 0 PM. Feedins and
waler management were h e same for all mamwnn.
Individual fish were measured every week for changes
i n body weight (BW) and body length IBLi.
Significant d ~ t f e r e ~inn the values between the
c o n m l and hormone mated groups w m dermlned
by DMRT afier A N O N .
3.1. Erpression. pun:fuation and choracteri-anon o i
recomhinanr cjGH
Thrce bacterial clones were found to express &H.
o f which expressed large amounts o f the protein
as judged from small-scale induct~onexpmimcnts.
NO
SDSPAGE detected a major band with a similar
mobilir). lo that of human GH (F:?. >A). \'estern
blot analysis revealed that this band reacted with an
antibody raised against native punfied rfGH which
had a similar mobility to that o f GH detecred in
pituil;u). exmcts o f the gilthead seabream (Sprur
o~rroro)(Fig. 2B). indicating that the clones indeed
produced recombinant f f i H protein. Furthermore, the
Western blot analysis indicated that antibodies nised
against the homologous native peptide recognized the
recombinant peptide produced in E coli.
IB conmining rtGH were prepared from -30 g wet
weight bacterial pellet obuined from 10 L as described in Materials and methods. An aliquot o f 100
p I was laken for analyzing the amount o f rfGH in the
I B by SDSPAGE using recomb in an^ bovine G H
(bGHI as the standard. The yield o f f f i H in the IB
was calculated to be -60 me. The protein contained in
Ihe I6 was refolded in 4.5 M urea as described in
Materials and methods. To determine the optimal
concentration o f cysteine needed for proper refolding.
the purified rfGH was refolded i n h e presence o f
several camlyric concentrations o f cysteine: 0.1 mM,
0.2 mU. and 1 mM.The refolded rfGH was purified
on a Q S e p h a w column by stepwise elution wirh
incmasing concentrations o f NaCl In 10 mU Tris pH
9. The purified rfGH. which was eluted i n I 0 0 mU
F t g 2 SDS-PAGE and W a v m Mm d ? ~ of
a lopl crlluLr
pmtetns from E roll cdlr Lane I. R r v r w a c x m hum Spvur
d u m ~ rlane
~ : 2. Clone n2 oipET &H. lane 3. rnwmbmull M H A.
C o u m s t c blue stalnnng: B. W a r m btof u ~ r ha n n w ntd
aqannw Mtrre punlied &H.
SaCl (Fig. IA). was anahzed by elecmphoress
u n d n non-reducing conditions and is shown in Fig
56. In all three cyxteine c o n c e n ~ i o n smed for
refolding. the purified rfGH appeared as a monomer.
Aliquols o f the peak fraclions, shown in Fig. 5.k were
analyzed for monomer content by gel filmicn
lhrough Superdex 75 column and wem found lo contain a monomenc rfGH (dam nor shown). The elution
profile obtained with a discontinuous -madimt o f SaCI
In 10 m\1 Tris pH S and in the presence o f 0.2 mS1
cysteine is shown in Fie. 4.4. .4nalysis o f aliquols bkSDS-PAGE with or without @maapmerhanol
re\.ealed monomeric rfGH (dam not shown). F m h a
analysis o f the peak fractions eluted uith SO n\l
SaCl and I 0 0 mu NaCl (shown in F i g 1A) for
monomer content by gel f i l u a ~ ~ orhrmgh
n
S175 column is shown in Fig 4B. The rfGH eluted i n -W
mu SaCl were almost pure monomeric form ullile
with 100 m.U KaCI impwities appeared. Companwn
o f elecmphoretic mobilin. o f rfGH eluted m 0 m\l
NaCl by gel electrophoresis with or withoul Ci-mercaptoe~hanol from a thii preparauon o f rfCH
revealed monomeric form. Furrhenmrr under mnreducing conditions the mobility uas s h i M slightly
forward. indicating a globular s n u m i F ~ g -4C)
Fractions h a t mntained monomeric pmuin s
shown by SDSP.4GE and HPLC anal)=
wur
p o o l 4 dialjzed against 0.05% ic'aHCOj and Iyophilized The monomeric smclwe and idenl~ryof ihc
dried-rauspended rfGH was furlher anal:&
by.
SDSPAGE under non-ducing condition. followed
by Westem blot. The ccsults. shown m F I ~5.4.
revealed over 98% pure band o f - ? I kDa. u-hich is
recognized by anti-native 6 H antibody (Fig SB).
The yield oblained from Z L o f E cuh fulm
was calculated as -5 mg o f pure m o m c &H.
The molar extinction u r f f i c i e n l al ZBl om was
calculated using the molar extinction o f I)nuwle
(1 197) and ryptophan (5559) yielding a kalue o f
II
, e n , 19hh). The spac~fic
for a
O.l?-6 solution (ms'ml) was 05-89. assuming a
molar mass o f 21.000 Da
3.2. Chmc~erirarion of onn-fiH
rjc~
~m~ mo,&;nanl
anrhodie b~
As s Ant step towards developing a quanoIatikx
assay (ELISA) for determination o f GH levels in
BEST AVAILABLE COPY
Tube m.
-
-
Fig 1 (.+I Elutm profile of reFomblnant ffiH WfKd on Q - S c p W mi-.
Tbc OScphmv m b ra e g u W d 10 mtt TmHCI pH 9 The pmvln
eluted r l l h a d ~ ~ o a m u x
gradnslr
s
of S K I in k ~m bullcr. of -%3M mM SdX Foi I ml rm
collated. The pmwin conrnmrian was mmmd by lbwrbvlo u ?80 m.(8)SDSPAGE ofprnhcd &H. dDLdcd urn
pacm at
dlllirsll conmumam of w n n c and clecaophoraed m ~ 1 5 %
gmd~rnls ~ ~ p o t ? m - k . dgrl
c I. ?bc
of 7
LI and 10. MU' d c r . Ian?.I mhf .ylc1nc(5 111): Lvrr 3 d i 0 I mM qtlrnnc(5 ard 10 )ill. Lurr 5 m d 6.6.2 mU 9%- ( I mt
I0 plb lams 7. 8 d 9. reccmbnmr ovme GH a 10. 20 lad 30 115The gel uar mrned wlh -C
B d h Blur R 2%
s e m o f rabbitfish. the antibody raised against mtive
purified f f i H (:\)son el AIL.30(W)) and the recombinant f f i H purified in the current swdy were charactenzed. The results o f the ELISA, summarized i n
Table I, showed concentrationdependent and dilutiondependent reaction. For f u w determinalions.
the antibody can be diluted benveen 1:40.000 and
1:100,000 and ELISA plates should be coaled with
I p_e ml o f pure recombinant f f i H , which gave 12.5
to 21.I P - 3 values The 10-fold dilution o f anlibodies
for ELISA compared to the dilution used ior \\&tern
*
blot (see Fig. SB)is mithin the accepted diludons u d
for rhae d~ffermt&says.
3.3. Assnvjor the b i o l q ~ m actirin
l
ofr~rombinant
r/GH
Ln an in bim e q x r i m e n ~liver uplants fmm rabbitfsh
were incubated in medium containing recombinant f f i H at doses o f 1-500 ng'ml for 24 h. A
slight but s~aristicallynot si@~ficanriin h e
levels o f ICF-I mRYA was noted in explan~sincubar-
BEST AVAILABLE COPY
" ,
0
10
20
30
40
Tube no.
50
60
70
B.
50 mM NaCl
I?==
524-xm
CHI
100 mM NaCl
S15-rm MI
a
FIE 4 ,A) E l u l m p o f i k o l d m t &H plnficd on QSrphamv c o h m T h i QSqbmemh.nn wm&hb.ad
nlb 10 m
u TrsHCI pH 8 The w o n u-rr e l u d wrh a darunnnuur g d m l o l NaCl m rhe ramc bufkr. of 50-?00 mM S K I -F
or I ml *a
mllec:ed Thi polm co-IROM
mild by a k ~ c b m a
t 280 nm (Bi Lkmmmlm of ihc momma C:of mH b? &
filna~~on
chrnmmgrqhy an a Sup&.
75 m h . RJr karuunr fmm Q ScphmY i o h cluoed n&50 mM SKI a d lW d
lY L l
rhoun in .A. mpecavrly Thi nvmkrr above rhe p s k rhow IIhc mmlion mm m m u l c r fCi SDEPAGE d r u ol &TjH d w d I. -X
m\l naCl irasloon. Lmr I. \I\\. marker lam I. ~T*lmiwn.i?du~tag
i~dnimm( u ~ i b.I-i ?
LUY 5. & ~ v d m
I&
p-nxrraptcdhanol) The gel u= rlrsned ~ 8 t hC,r,mairlc Bnllranr Blvc K 2 i i l ~
:*
BEST AVAILABLE COPY
Fd_e 5. SDSP.4GE and W e r m b l a a n a l p i of pure rrxmbml &H. lam I. CNdc pepmoo. of
boma
2. h k d
p-tlon
af~nclusionbodis; lux 3. Rm &H ~ f o l d c dIn the -c
010.2 mu w
u
r and dud m l lm n\l ?i.CI lac L.g L:
e l c c m @ o d wnhout ~mmaprurhual;15. Ruc f f i H d i d > m l 10.05% SaHCOj. I!ophtlual a d V cli S d c HQ.
elcc?mphaacd wilhout &-macaptahaml; bne 6. Raombmmt o v m GH (10 MI (A) C o m m s t c B l u samms I B i ua;m Nx
~mmwurcactedr t r h mtr-native &H antlkdy
ed with I00 ng'ml for 24 h (Fie. 6). Other doses were
not effective in stimulating IGF-I gene expression in
the liver explants compared 10 the controls.
In vivo, however, a significan~increase in BW
gain was o b m e d starting on the 2nd week in
rabbitfish fry injec~ed with the recombinant f f i H
a1 a dose of 0.5 p g g BW compared to the control
and those injected with 0.1 and 0.01 p g g BW (Ftg.
'A). This significant increase in the BW gain was
observed until the 4th week of the experiment. No
significanl difference in BW gain was oberved
berueen the fry in ihe control p u p and those
mated with 0.01 and 0. I p g g BW during the entire
duration of the experiment. Similarly, a significant
SWO
10.000
Z0.W
4O.W
so.m
IMl.WO
0
>4?
>42
>42
>42
>42
>42
33 4
30 1
I .
18.1
17.7
I
1.0
1.0
>42
>4Z
>42
U.5
0.2
IS.?
1.0
W.0
24 5
I4 I
I
81
I
8.2
28
I
47
4.2
10
zo
80
4.5
2.7
1.7
1.2
17
1.0
1.0
>42
>4Z
40.4
12.5
10
4.5
increase in body length WBS observed m @ tnjmed
with 0.5 pg'g BW aRer the 2nd week; this *as no1
o b s m e d in those that w m inuith ihe lou-er
doses and the control ( F I ~-B).
In the presenl srudy, we rep^ on t
k crprnrian,
refolding and purifidon of recambinant f f i H .
which is conlainad in inclusion bodia. Thc yeld
war -5 mg of close 10 98% pure moanncric &H
fmm 2 L of bacterial culrurc. The pollam
found in the 'ac~ion eluted nith 50 mW SaCI iuhcn
1.1
-
The optical denrmry (OD) u.%sd+rcrm~ncduang a m E L I S A
reader at 405 nm. Each ELlSA -11
*rzr c r p d a Ihc w o
berwcen the mean absorbanre of tested ramplc in pmaorc IPI and
the mean rbsorbancc in Depmc 1x1 wells.
-y
u x per-
h g 6 IGF I m R t 4 b c k LI .1&h
11,s d r a mo&ad m
wtrm m m d u m c m r a ~ g
mnrmmmm o i
W*If i H IFF-I Y*l Frnn pcne crvm c k m m d
f o d in mpllcats.
hb
RT-PCR
BEST A V A I U B U COPY
A
+0l~ggBW
0
1
2
3
weeks
4
5
B
c
20 7- 0 01 pgfg
BW
s
0
0
1
2
3
weeks
4
5
Fug 7 Thc e f f m or r o o D m b l ~ n 1f i H on l"cmaw m bod, *rnght
I A l dI d kagm ( B Iolrabb~rfishfry a h w d l ) h n r m a n c m
ofrhc h o r n Thc .auk M pevnrcd a -I
VJua w ~ l h
atensk art sngnnfiirly dnff-I
imm rhc control
...
the refolding was carried out in 10 m\1 Tris, pH 8) but
in the 100 mll KaCI fractions when the refolding was
carried out in 10 mM Tris, pH 9. .4nalysis by HPLC
and SDSP.4GE under non-reducing conditions
revealed a monomeric form. indicating a c o m t
refolding of the polypeptide under both pmtocols.
Furthermore, comparison of the mobility of the purified refolded f f i H by SDSPAGE under reducing and
non-reducing conditions showed a slight shift forward
in the mobility in the absence or pmercaptoethanol.
Even after lyophilization and reupcnsion. the peptide retained its monomeric form. The protocol for IB
preparation. refolding and purification of rtGH followed essentially that described for S. awara GH
IsaGH. B e n - A l i ~et 3i.. IQW). although in the laner
repon an additional protein (placramsse) was coproduced. co-purified and co-eluted with saCH by
100 dl NaCI. The recombinant f f i H WTS tested
for its suitability for the development o i m ELISA.
using polyclonal a n u i i e s raised in rabbits against
the purified native f f i H (4ywr: et 31. r)iJnl. which
was also used for Wenern b l o ~to v m @ the identir?.o i
the expressed ffiH. The results of the ELIS.4 demonstrated a high titer for the antibdies. based on their
ability to detect recombinant f f i H at \-- IOU- concentrations (0.01 pg'ml) when used in a high dtluhon
( 1:80.000).
Although numemus sndia ha\.e k e n published
on the expression and purification of recumbinant tish
GH. in this paper we describe the refolding of a fish
GH that carries unusual features compared IO o h
fish GHs (.Aywn et al.. 10(X)). Rabbitfsh GH is larger
by 5 amino acids than the flounder GH. uhich is the
smallest teleon GH (173 amino acids) makine it h e
second smallat GH molecule. It has a unquc characteristic: 5 amino acid deletion in paritions I4SI47.
immediately preceding the I4 amino acid deletim m
flounder GH. In addition, it has single a double
amino acid deletions. This deletion of 5 amino aciris
is unique to rabbifih GH. These 5 amino a-ids am
even present in flounder GH. At this point we do nol
know the biological significance of this dcktion. Despite this f e a t ~our
~ , data suggest that the peptide uas
folded correctly, using the proroml dccailed in the
ma nu scrip^ In addition. it could be argued lhat the
inuinsic activity of GH is IOU- in rabb~rfirhand t h ~ s
probably explains the slow groulh of f a b b ~ d s h(pmb
ably one of the factors). In order IO pnaw thrs unique
chvacteristic of rabbitfish GH. it baame n e e s a y ar
a f i s t step IO expms, purify and fold cum+
rabbitli~hGH.
The biological acn\.it). of the mombinaot rRjH
was tested both in \IUO and in \ivo by employing
homologous systems. In vivo. rabbirf~hfr). injected
intra-peritoneal (IP) with the recumbinant f f i H at a
dose of 0.5 pg'g BW already showed significant
increase in growth starting on the 2nd u.eek of treatment and thls u-as maintained as long as the hormone
injection was given (4 weeks). The lower doses (0.01
and 0.1 pg;g BW) of the hormone used did not eltcit a
faster growh. A slight difference in the bioadvir).
was found between the gmu+ expenmenu conducted
u1th the recombinant f f i H tlhis study) and our previous repon using the native f f i H that was purified
fmm the pimilary gland (A?wn ct 11.. ?(.<I)).\\hie in
in gmu*
our previous work a significant inwas already observed in f r j injected u-ith a dose of 0.1
pg's BW, this dose was withoul effect in the present
study. This difference can be amibuted to a lower
specific activity o f the recombinant rRiH. Although
by SDSPAGE and HPLC analysis the recombinant
rfCH appeared as a monomer, the amount o f purified
protein wed i n the nvo systems could have been overor under-estimated, depending on the way the amount
was calculated. Nevertheless, a positive effect o f the
recombinant rRiH on growth, even with a higher
dose. can be considered as an indication for the b i e
logical activity o f the refolded, purified f i H . Similar
results were reported for several other fish species. in
which an increased growlh was also observed following injecuon o f recombinant fish GH. Thw, in mullet
(.\tug;/ cepholur), fish receiving tP injections o f recombinant porgy G H every 2 weeks for 16 weeks al a
dose o f 0.1 p p _e B W showed significant increase in
gmw-th. starting on the 6th week with a lesser effect
seen in those injected with a higher dose o f I p g g
B W and no effect was seen when a high dose o f 2 pg'
g B W was used (Tsai ct at.. 9 9 ello on et 31.
I I')SR:I also reported increased growth rap in rainbow
trout injected with recombinant h u t GH at a dose o f
0.2 p g g BLV. Likewise, slower growth was observed
in those injected with 2 pp'g BM'. In the present study.
we did not use a dose higher than 0.5 pg'g BH_
Slightly different results were found i n sea b m
juveniles. i n which a dose o f 2 pg'g EN' eliciled an
increase i n growth rare (Bell-Ati:! r t al., IWU).
By contrast. the i n v i m experiments for testing the
biological activity were less promising as only a
m a ~ i n a effect
l
o f the recombinant rfCH on IGF-I
eene expression in liver explanls tiom rabbidish Fry
was noted after a 24-h incubation with 100 n p m l o f
the hormone. I n previous studies. primary cultures o f
hepatocytes, rather than tissue explants, were used for
testing G H activity. Thus, Duan et al. ilo9;l observed
a dosedependent increase in IGF-I m W . 4 levels in
coho salmon a k r incubation with native coho salmon
GH at a dose ranging fmm 0.01 to I p g m l . Sharnblon
sf JI. l19')51 also observed an increase in IGF-I
f i . 4 levels i n primary cultures o f hepatocytes in
rainbow trout after incubation with bovine G H at
similar doses with that o f Duan
al. I 1Q9.il. X
dosedependent increase i n IGF-1 mRNA determined
by RT-PCR was reported also in p r i m q culrures o f
tilapia hepatocy-tes cultured in the presence o f recombinant tilapia GH (Scl1m131LA 31.. 20011). However in
-
the laner study no correction *as nudr to a conwltutive &%A
like Factin. for example. and therciore
the apparent effect could be over-shrnated A daudependent inin IGF-I mRSA levels i n thc l i b e r
o f coho salmon was also o b e d after a slngle LP
injection o f coho salmon GH ( h u r c: a:.. 109:).
Several possible explanations could be o f f d i;u
the lack o f significant induction o f IGF-I mRXA in
liver explanls and Ihe d i f f e m b e e m h e m vivo
and in v i m assays. Smce i n the c m t study ur uud
rwo different preparations for thc in b i ~ v
and m w m
bioassays, bariabiliv in the potency o f the nr-o prrparations or inaccurale d e t e r m i d o n o f pmein concentration could be one reason for low aclivit). In the
in v i m experiments. Another pcssibility is poor pcnetration o f the r f c H lo Ihe tissue explants. Sum Ihe
[issue explants were maina~nedin Ihe a b m x o f
serum or other pmtein substitution. 11 could have
affected also the integrity o f he tl+aw although this
is unlikely since bypan blue exclusion tra of thc
tissues showed ne-mive results.
Numerous srudies repolled on poduwn o f recombinant fish GH pmltins. Exccpl for few cavr
where the pmtein w w p r o d u d in a a - o b c expression sptem (TSI el al . :W; Ho d a!.. IQQ*).
most o f tbese mornbinant fi+h GHs hd been
expressed in bacterial cells (rebxU.ed in Fmkmdrcr..
2MN) Polypeptide expression i n bacrenal cells is Ihe
method o f choice for the production o f simpk pwcins
ltke GH that needs no p o s t - m s l a M m o d ~ t i a a o m
because i t is easv. m o v e r is fas~s i r n b a u r r ~arc
-mnn for only a day. and the yield is hi&. S i m h to
carp. sea bream and dolphin fish rccombinant GHs.
rfCH also conmined an a m i n e m i n a l mcchionine
residue. Thc law nucleotide donaud by the .Vml
resuiction site (uud as one o f the cloning sim in
the p m d vector used in the cumnl smdy) a d h e
sequence of the fim amino acid residue o i t h e nwum
f f i H made 11 m x s a r y to add N;O nuckotidcs m
order lo keep h e reading frame. t b u creating an
additional codon for tbe amino acid alanine. R e v ~ o u
studies have shown IJW i n some casa the additional
amino-terminal methionine is almost completely
being processed and he pmltin is alanyIGH. ltke in
the case o f S aurora G H (Ben-.Atla 31. IUW) while
in other cavs the additional methion~ne no1 cleaved
durine processing by E coli. l ~ k ein Ihe case c f
dolphin fish I P n d ~ c l e: n l . :MI. I n Ihe sre o f
c v l r w 250 12W5i SW-515
carp GH, about IZ-13% were Met-GH (Fine e l al..
14951 The three preparations, which were folded
using a similar protocol to that described in the current
study, retained their biological activity in several
assays.
There is an increasing interest in the application o f
G H therapy in fish in aquaculture since this possesses
the potential to increase fish production. While there
are several ways to administer the hormone IO the tish
(injection, immersion), incorporating the protein in the
diet is the most practical way for its mass application.
T h i s method, however. will become etTective only if
fundamental questions surrounding the method like,
avoidance of enzymatic degradation in the gastrointestinal bact, quantification of the hormone concentralion in the feeds, efficient mixing o f the protein in
the feeds without losing its activity, among others, can
be addressed effectively.
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We thank Dr Sara ~Uauriceo f the Hebrew Universin. of Jerusalem for assistance in recombinant G H
purification. We also thank Fiona Pedrmo. Edgel May
Bayag, Hannah Cordero and the staff o f the Fish
Hatchery of SEAFDEC AQD for assistance in various
aspects of the project. This work is supported by a
grant from US.UD (Grant No. TA-MOU-99-C 1940I.
Program in USlsrael Cooperative Development Research. Economic Growh).
References
.igclbn. L.B.. E-.
CJ.. J-.
J.M.. D a v k S.L.. h g k . A.D.
TT. 1988. Rwnodun of npd w u l h of mnbou UaoI
j S d m gurmhm) by a raromhmc fah p u l h h m n e Can.
J Flrh Aqua El.41. I*-151.
.A,pon. FG. dr JE.G.T. A m i s Y . >tonyS.. H m .
T. hwauchn. H . IPYP lvlbtan imd cDS.i cbntng of mtolaxm m &bluish !Signnu g u m 1 Gm. Comp. Edc-znml. l IS. 292-300.
i j w n , F.G. & Jculr. E.G.T. . h y a Y. M o n y m u 5 . HI-.
T. Kawrrh~.H.. 2aT). hlarim cDNA clonrng imd gruulh
p m i n g acnr-~;yof rabbidlrh ( S i p w gumnu) w u 7 h bmnc G m C o w . Erdocmol 117. 3 1 -259~
A!ron. FG.. de Jslri, E G T.. M o t i ~ w
S . Hyodo. S.. F d m stein. B.. Gmla.
hwauchr. H.. 2002. Dnllmual a p r a rwn or inrul~n-liic grouih b a o r I d 11 rnR5.Q dunnp
..
KT.. D l s s S . O r . A . P K . L q J . ~ W M . % \ ~ .
Wong A.OL~. 199? hpcmca o l g p r cmp m b
by -nna
m
h r El.Khm Ek#B.rr .is
1381. 331-33%
Lam TJ. 1974 Sagvudr a d dm mancuhm
.Aapxd
rn 3. 3U-3.S
M*
H., -r
K.. %C.. L ~ o m
T. Yobpo. T. IW
The Flrher o r k- J
. b j q e @ m T&
CM-~
Raa
J a w 437 p p ~
MotiyS.. TJ;lutr T. Y a m q d u . K . b& H .
M o h k c h m g of d m Ikc ~
- f w chD S A s rd b
uprcrwn la Jlpaaac scl Fah Sa 68 tSlpp( rr 5 " - - 5
MwI. A.. Clupn+Cohe4 5 . Gsrb. A. Ellop. I . 1999
R e p u a t k d charmrnnan of mamkew AaW= Wi
(C-pkem h,-I
p 7 h hmma Rm b p Ant' 14
11--423
Pnttn\.SJ.1986 R a c l . v o h n n s m l h y h 5 m & t . J
E l l o c h . IS?. 169-180
W m d . A.C. R&+
H . KLoa. U-.
Nh.al
heptqm or k bm? fah. am~ T Y Idr
~ P .
ubpu a rdd :ml i c r p h + a ~adp a IGF-I
m lur= J Eodoouol 166. 265-?i3
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