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cDNA sequence analysis, recombinant expression ofthe inhibin α-subunit of the yak (Bos grunniens)Hong-Mei Wanga, Xiang-Dong Zia & Hong Zhoub

a Key Laboratory of Animal Genetics and Breeding, State Ethnic Affairs Commission andMinistry of Education, Southwest University for Nationalities, Chengdu, PR Chinab The Hospital of Southwest University for Nationalities, Chengdu, PR ChinaPublished online: 29 Jan 2013.

To cite this article: Hong-Mei Wang, Xiang-Dong Zi & Hong Zhou (2013) cDNA sequence analysis, recombinantexpression of the inhibin α-subunit of the yak (Bos grunniens), Journal of Applied Animal Research, 41:3, 255-262, DOI:10.1080/09712119.2012.742440

To link to this article: http://dx.doi.org/10.1080/09712119.2012.742440

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cDNA sequence analysis, recombinant expression of the inhibin a-subunit of the yak (Bosgrunniens)

Hong-Mei Wanga, Xiang-Dong Zia* and Hong Zhoub

aKey Laboratory of Animal Genetics and Breeding, State Ethnic Affairs Commission and Ministry of Education, SouthwestUniversity for Nationalities, Chengdu, PR China; bThe Hospital of Southwest University for Nationalities, Chengdu, PR China

(Received 6 June 2012; final version received 15 October 2012)

Inhibin plays an important role in the regulation of reproduction in mammalian species. The objectives of thisstudy were to analyse cDNA sequence and produce recombinant yak inhibin a-subunit protein in Escherichia coliby optimising the expression conditions. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy,we obtained full-length yak inhibin a-subunit cDNA sequence comprised of an open reading frame of 1080 bpencoding a 360-amino acid protein with a predicted molecular mass of 38.8 kDa. The predicted protein sequenceincludes 4 possible proteolytic cleavage sites, 12 evolutionarily conserved cysteine residues and 2 potentialN-linked glycosylation sites. The mature subunit is the carboxyl terminal fragment (C-fragment) consisting of134 amino acids. The full-length precursor protein shared 79.6�98.6% protein sequence identity with mammalianhomologues. It exhibited the highest identity with that of cattle (98.6%); there were five amino acid substitutionsbetween these two species. The homology is not evenly distributed with the highest level for the putativeC-fragment (77.1�99.3%). The recombinant yak inhibin a-subunit protein was successfully expressed in E. colifor the first time.

Keywords: inhibin a-subunit; cDNA cloning; sequence analysis; expression in E. coli; Bos grunniens

1. Introduction

The mammalian reproductive cycle is regulatedthrough the actions and interactions of hormonessecreted by hypothalamus, pituitary and gonads(Woodruff et al. 1987). Follicular development andspermatogenesis are dependent primarily on theactions of follicle-stimulating hormone (FSH) andluteinising hormone (Steinberger 1971; Richards et al.1986). Inhibin, primarily produced by ovarian granu-losa cells (Erickson and Hsueh 1978) and by Sertolicells of the testis (Steinberger and Steinberger 1976), isa glycoprotein hormone originally purified fromovarian follicular fluid (Ling et al. 1985; Miyamotoet al. 1985; Robertson et al. 1985). It selectivelyinhibits FSH secretion (Steinberger and Steinberger1976; Schwartz and Channing 1977; Kaneko et al.1997) and FSH action by suppressing FSH receptorexpression on granulosa cells (Lu et al. 2009). Inaddition to its action on FSH through hypothalamo�hypophyseal�ovarian axis, inhibin may also be play-ing an important role in regulation of folliculogenesisthrough autocrine and paracrine control (Findlay1993; Campbell and Baird 2001; Bhardwaj et al.2012a). It is composed of an a-subunit linked witheither a bA or bB subunits by disulphide bonds toform active dimers known as inhibin A and inhibin B,

respectively (Mason et al. 1985; Campbell and Baird2001). Inhibin a-subunit precursor cDNA sequencesfor many eutherian mammalian species have beenpublished, such as porcine (Mason et al. 1985), human(Mayo et al. 1986), bovine (Forage et al. 1986;Bhardwaj et al. 2012b), rat (Woodruff et al. 1987),murine (Albano et al. 1993), equine (Yamanouchiet al. 1995) and possum (Vanmontfort et al. 1998). Thefunctions of inhibin suggest a role for the substancesleading to suppression of endogenous inhibin or itsderivatives and other agents regulating its production,as a fertility-augmenting substance in the case of farmanimals and small ruminants for increasing thenumber of ovulations and offspring. In agreementwith this idea, passive or active immunisation againsta recombinant a-subunit inhibin has been shown toincrease ovulation rate and fertility in sheep (Han et al.2008; Naqvi et al. 2009; Guo et al. 2010), goat (Padillaet al. 2008; Wang et al. 2009), gilts (Brown et al. 1990),mares (Mckinnon et al. 1992), hens (Satterlee et al.2002) and cattle (Li et al. 2009). Therefore, manysystems for heterologous inhibin production havebeen developed, but the Gram-negative bacteriumEscherichia coli remains one of the most attractivechoices because of its ability to grow rapidly andat high density on inexpensive substrates and the

*Corresponding author. Email: zixd2000@yahoo.com

Journal of Applied Animal Research, 2013

Vol. 41, No. 3, 255�262, http://dx.doi.org/10.1080/09712119.2012.742440

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availability of an increasingly large number of cloningvectors and mutant host strains (Bhardwaj et al.2012a, 2012b).

The yak (Bos grunniens) is adapted to the chronichypoxia of high altitude (�3500�5500 m) and is themost important farm animal species in Qinghai-Tibetplateau of China by providing meat and milk wherefew other animals will survive. However, the femaleyaks have low reproductive rate (40�60%; Wieneret al. 2003; Zi 2003), and comparatively little isknown about the structure and function of inhibinsin yaks. Inhibin plays an important role in theregulation of reproduction in other mammalianspecies, therefore, the objectives of this study were(1) to clone and sequence the yak inhibin a-subunitand (2) to produce recombinant yak inhibin a-subunitprotein in E. coli by optimising the expressionconditions.

2. Materials and methods

2.1. Animals and sample collection

All experimental procedures were performed accord-ing to the guide for animal care and use of laboratoryanimals of the Institutional Animal Care and UseCommittee of Southwest University for Nationalities.Five adult female Maiwa yaks used in this study weregrazed on natural grassland at a height of 3200�4000m in Hongyuan County located in Qinghai-TibetPlateau, Sichuan Province of China. They weretransported and slaughtered in Xiangyang slaughter-house, Chengdu. Ovaries were collected and immedi-ately frozen in liquid nitrogen and stored at�808Cto be used for RNA extraction.

2.2. RNA isolation

Total RNA was extracted from whole ovary of theyak with RNAprep pure Tissue Kit (Tiangen, Beijing)following the manufacturer’s instructions. The RNAswere quantified using the spectrophotometer (Eppen-dorf, Germany), and RNA integrity was evaluated ona 1% (w/v) denaturing agarose gel. Only RNAs withA260 nm/A280 nm ratios of 1.6:2.0 were used for reversetranscription-polymerase chain reaction (RT-PCR).All samples were stored at�808C until cDNAsynthesis.

2.3. Oligonucleotide design

Oligonucleotide primers for the amplification ofyak inhibin a-subunit were designed based on theconserved region of cattle inhibin a-subunit(NM_174094) using primer 5. The forward primerwas 5?CCCAAGCTTCAGGGGAGCTATGTGG-

CTTCAG3? (HindIII recognition site is underlined)and the reverse primer was 5?CGGGATCC-CACTATGTCATTTATTGTCACAGCGG3? (Bam-HI recognition site is underlined). The primers weresynthesised by TaKaRa Biotechnology (Dalian) Co.,Ltd.

2.4. RT-PCR and sequence analysis

Reverse transcription was performed using RNAPCR kit (AMV) (TaKaRa, Dalian, China) in a totalvolume of 10 mL reaction mixture, which contains 0.5mg RNA, 5 mM MgCl2, 1�RT buffer, 1 mL dNTPmixture, 10 U RNase inhibitor, 2.5 U AMV-RT,1.25 pmol oligo(dT) primer and 3.75 mL RNase freedH2O, at 458C for 45 min, 998C for 5 min, one cycleat 58C. The generated cDNA was then amplified in atotal volume of 25 mL of reaction mixture containing2 mM MgCl2, 1�PCR buffer, 2-mM dNTP mixture,20 pmol each primer, 15.5 mL RNase free water, 2.5 UEX Taq HS and 0.5 mL cDNA. Thermal cyclingconditions were initial activation at 958C for 1 min,followed by 35 PCR cycles of amplification under thefollowing conditions: denaturation at 958C for 40 s,annealing at 588C for 40 s, extension at 728C for 90 sand a final extension at 728C for 5 min. PCRproducts were separated by agarose gel electrophor-esis, and the band of desired size was excised andpurified using AxyPrep DNA Gel Extraction Kit(Axygen Biosciences, CA), and subsequently thepurified DNA fragment with desired size was clonedinto T-vector [TaKaRa Biotechnology (Dalian) Co.,Ltd.] and transformed into DH5a (E. coli) usingstandard techniques (Sambrook and Russell 2001).The DNA sequence was determined with an ABIPRISM 3700 automated DNA sequencer. The se-quence obtained was subjected to BLAST analysis(http://www.ncbi.nlm.nih.gov/BLAST) to verify thatthe sequence was of inhibin a-subunit. Nucleotidesequence identity was performed using Clustal pro-gramme of MegAlign (Lasergene software, DNAS-TAR). Alignment of deduced amino acid sequencewas also carried out by Clustal multiple sequencealignment programme. The signal peptide was pre-dicted by Signalp 4.0 (http://www.cbs.dtu.dk/services/SignalP/). The potential glycosylation sitesand proteolytic cleavage sites were predicted byNetNGlyc 1.0 (http://www.cbs.dtu.dk/services/) andExPASy (http://www.expasy.org/), respectively.

2.5. Construction of yak inhibin a-subunit expressionvector

The PCR product was purified using AxyPrep DNAGel Extraction Kit, and digested with both BamHI

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and HindIII. The digested cDNA was inserted intopET32a(�) expression vector that had been digestedwith the same restriction enzymes. The construct wastransformed into E. coli BL21 (DE3), and the cellswere grown in plates with Luria-Bertani (LB) andagar medium containing 100 mg/mL ampicillin. Thepositive clones were further selected by PCR screen-ing. Subsequently, the plasmids were sequenced toconfirm there was no alteration of the coding domain.

2.6. Yak inhibin a-subunit recombinant expression

A single colony harbouring the plasmid was pickedand incubated in 5 mL of LB medium with 100 mg/mLampicillin at 378C overnight. Six test tubes weremarked, respectively. In each test tube, 0.1-mL over-night culture was added to 20-mL prewarmed LBmedium, which was shaken at 328C until the OD600

was 0.518, then the isopropyl-b-D-thiogalactoside(IPTG) was added to a final concentration of1.0 mmol/L. A time course study of expression wasperformed to determine the time on the production ofrecombinant protein; after induction for 0 (no induc-tion), 2, 4, 6, 8 h, 1 mL samples of the culture in eachtest tube were taken, the cells were harvested bycentrifugation (12000g for 10 min) and stored at�208C after supernatants were discarded. Subsequently,the samples were dissolved in 100 mL of loading bufferand boiled for 5 min, centrifugated (12000g for 10min) and then a 10 mL of supernatants was loaded on a10% sodium dodecylsulphate�polyacrylamide gelelectrophoresis (SDS�PAGE) with low range mole-cular weight markers as molecular weight markers[TaKaRa Biotechnology (Dalian) Co., Ltd.].

3. Results

3.1. Molecular cloning of yak inhibin a-subunit cDNA

By RT-PCR strategy and determination of the DNAsequence, we obtained the yak inhibin a-subunitcDNA nucleotide and the corresponding predictedamino acid sequence (Figure 1). The coding region ofyak inhibin a-subunit cDNA (1080 bp) encoded a360-amino acid precursor protein with a predictedmolecular mass of 38.8 kDa. This sequence of yakinhibin a-subunit has been submitted to GenBank(JN572112).

3.2. Nucleotide and amino acid sequence identity

The nucleotide sequence as well as deduced aminoacid sequence of yak inhibin a-subunit were alignedwith six eutherian homologous sequences (Figure 2),and the degree of identity is presented in Table 1. Itshowed that the deduced amino acid sequence

identities were 79.6�98.6% for full-length precursorprotein, 73.8�98.8% for N-terminal fragment and77.1�99.3% for C-terminal fragment.

The inhibin a-subunits showed that the proposedsite of signal peptide cleavage was assigned betweenGly17 and Cys18, and three evolutionary conservedproteolytic processing sites were identified at Arg59�Arg60, Arg68�Arg69 and Arg225�Arg226 (Figures 1 and2). On the basis of homologies with mammalianinhibin a-subunit, the a-subunit proinhibin is pre-dicted to be derived from its precursor by cleavage atArg59�Arg60, a common site for proteolytic proces-sing of precursor protein. The cleavage at Arg225�Arg226 generated two fragments of 166 (N-terminal;61�226) and 134 (C-terminal; 227�360) amino acids,respectively. The C-terminal fragment is presumablythe mature a-chain with the predicted molecular massof 14.65 kDa. Two potential glycosylation sites werefound in the a-chain. One is in the N-terminalfragment at residue 140, and the other is in themature inhibin a-chain at residue 262. There are12 cysteine residues in the whole a-subunit precursor,of which one is in the signal peptide region (18), four(91, 194, 197 and 199) are in the N-terminal fragmentand seven (256, 285, 289, 321, 322, 357 and 359) are inthe C-terminal fragment (Figures 1 and 2).

3.3. Expression of yak inhibin a-subunit in E. coli

The yak inhibin a-subunit protein was expressed inE. coli (BL21) after cloning the cDNA into theBamHI/HindIII restriction sites of pET32a(�) ex-pression vector. The molecular weight of the obtainedfusion protein (59.4 kDa) is in agreement with theexpected 20.6 kDa for pET32a(�) and 38.76 kDa forinhibin a-subunit. Time course expression from 0 to24 h showed that after 4 h induction of therecombinant inhibin a-subunit protein was alreadymaximal (Figure 3).

4. Discussion

In this study we isolated cDNA clones encodingthe ovarian inhibin a-subunit molecule of the yak(B. grunniens). The nucleotide sequence was deter-mined and amino acid sequences were deduced. Tothe best of our knowledge this is the first study toreport the entire nucleotide and deduced amino acidcomposition of ovarian inhibin a-subunit of the yak.The coding sequence of yak inhibin a-subunit com-prised 1080 bp open reading frame (ORF) whichencodes a 360-amino acid precursor protein contain-ing a signal peptide of 17 amino acids (1�17), apro-fragment of 43 amino acids (18�60) andtwo fragments of 166 (N-terminal; 61�226) and 134

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(C-terminal; 227�360). The recombinant yak inhibin

a-subunit protein was also successfully expressed in

E. coli for the first time.It seems that the major functional groups of the

amino acid sequence have been conserved during

evolution, including the positions of proteolytic

cleavage processing sites, N-linked glycosylation sites

and the number and positions of cysteine residues

(Zhu et al. 2010). In eutherians, inhibin a-subunit issynthesised as a precursor protein that is processed by

sequential proteolysis, first removing the 17 aminoacids’ signal sequence (Forage et al. 1986; Mayo et al.

1986), and then cleavage at three dibasic sequences

producing various possible polypeptides of differentlength, of which the C-terminal fragment is considered

the mature a-subunit (Mason et al. 1985; Forage et al.

1986; Woodruff et al. 1987; Albano et al. 1993;Yamanouchi et al. 1995). In the present study, we

found that in yaks, this mature inhibin a-subunitcontains 134 amino acids with the predicted molecular

Figure 1. Nucleotide (cDNA) and deduced amino acid sequences of yak inhibin a-subunit gene. The mature inhibin a-subunit(aC) sequence is underlined. The positions of potential N-linked glycosylation sites (*), cysteine residues (�) and potentialproteolytic cleavage sites (') are indicated, as is the termination codon (***)(Genbank Accession Number: JN572112).

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mass 14.65 kDa, similar to those of the mature

a-subunit in porcine (Mason et al. 1985), bovine

(Forage et al. 1986) and equine (Yamanouchi et al.

1995) species. Like all other known mature inhibin

a-chains of all mammalian, the mature yak inhibin

a-subunit possesses a potential glycosylation site at a

similar position in molecule (36 of 134). The number

and distribution of cysteine at residues in the amino

acid sequences are also evolutionarily conserved

between yak and eutherian species (Figure 2). There

are four cysteine residues in the N-terminal fragment

of the amino acid sequence, suggesting possible

intrachain disulphide bonds that enable the separa-

tion of the N-terminal fragment from the C-terminal

fragment (Wang and Johnson 1993). The odd number

(seven) of cysteine residues in the C-terminal fragment

suggests that at least one cysteine may be free to form

a disulphide bond with the inhibin b-subunit duringheterodimer formation (Wang and Johnson 1993;

Vanmontfort et al. 1998).Comparison of the predicted amino acid sequence

of inhibin a-subunit from yak with that of other

Figure 2. Comparison of the deduced amino acid sequence of yak inhibin a-subunit precursor protein with six eutherianmammal homologues [bovine (NM_174094), buffalo (EU884446), equine (D50327), porcine (M13980.1), human (NM-002191) and mouse (X69618)]. Positions at which sequences are identical with that of the yak are indicated by dots. Spaceinsertions are indicated by �. The positions of conserved cysteine residues (�), potential N-glycosylation sites (*) andproteolytic cleavage sites (%) are indicated. The length for each protein sequence is given at the end of each sequence.

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species (Table 1) showed the highest identity withthat of cattle (98.6%); there were five amino acid

substitutions between these two species (Figure 2).Similar to the previous reports in other animal species(Woodruff et al. 1987; Vanmontfort et al. 1998), the

amino acid identity of yak a-inhibin for theC-terminal fragment with the six eutherian a-inhibinsequences (77.1�99.3%) is greater than the identity

for the N-terminal fragment (73.8�98.8%). Thecomparatively high level of sequence conservation

of the C-terminal fragment might indicate that it isthe dominant form of the different cleavage products,which forms in combination with a b-subunit biolo-gically active inhibin (Woodruff et al. 1987).

At present, the importance of inhibin on the

regulation of reproduction in mammalian specieshas been demonstrated (Steinberger and Steinberger

1976; Schwartz and Channing 1977; Findlay 1993;Kaneko et al. 1997; Campbell and Baird 2001; Luet al. 2009). The applications of recombinant a-subunit inhibin as an immunisation agent to increaseovulation rate and fertility have been an active area ofresearch (Han et al. 2008; Padilla et al. 2008; Li et al.

2009; Naqvi et al. 2009; Wang et al. 2009; Bhardwaj

et al. 2012a, 2012b). In the present study, the

recombinant yak inhibin a-subunit protein wassuccessfully expressed in E. coli for the first time.

This will provide a valuable tool for studying the

functional role of inhibin in the yak reproduction,although the purification and biological activity of

the recombinant inhibin a-subunit will be needed to

be further studied.In conclusion, the cDNA encoding the inhibin a-

subunit was cloned, sequenced and characterised

from the ovary of the yak, and inhibin a-subunitcDNA encodes a predicted precursor protein of

360-amino acids. It indicates considerable conserva-

tion of the structure and possible function of theinhibin a-subunit protein.

Acknowledgements

This research was funded by Southwest University forNationalities (12NZYTH07; 2011XWD-S0905); NationalKey Technology R&D Program (2012BAD13B06); SichuanProvincial Foundation for Leaders of Disciplines in Scienceand Technology, PR China.

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Figure 3. Expression of yak inhibin a-subunit protein inE. coli (BL21). Cells were cultured in LB medium with100-mg/mL ampicillin at 378C until the OD600 reached0.518. Then, IPTG was added to a final concentration of1.0 mmol/L. The cells were harvested after 0, 2, 4, 6, 8, 24 h(lanes 3�8) for induction of yak inhibin a-subunit proteinsynthesis, respectively, and total proteins were analysed by10% SDS�PAGE. Lane 1 is pET32a without induction,and lane 2 is overnight induction of pET32a.

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