Expression of bovine activin-A and inhibin-A in recombinant baculovirus-infected Spodoptera...

Expression of bovine activin-A and inhibin-A inrecombinant baculovirus-infected Spodoptera frugiperda

Sf 21 insect cells

CiaraÂ n N. Cronin 1, Devon A. Thompson 2, Finian Martin *

Biotechnology Centre and Department of Pharmacology, University College Dublin, Bel®eld, Dublin 4, Ireland

Received 22 January 1998; accepted 2 June 1998

Abstract

Currently, bioactive activin and inhibin for investigative purposes are obtained either by puri®cation from bovineor porcine follicular ¯uid or have been kindly supplied in limited amounts by Genentech. The latter arerecombinant formulations produced in cultured monkey kidney CV-1 cells. The aims of this study were to assess the

potential of the baculovirus expression system as an alternative means to produce recombinant activin and inhibin.Towards these goals, two recombinant baculoviruses, AcBovACTA and AcBovINHA, were constructed.AcBovACTA contains a contiguous copy of the bovine bA-inhibin/activin structural gene encoding the bA-preproprotein whereas AcBovINHA contains contiguous copies of the bovine a-inhibin and bA-inhibin/activinstructural genes encoding the a- and bA-preproproteins, respectively. Western blot analyses, using monoclonalantibodies speci®c for the mature portions of the a-inhibin and bA-inhibin/activin subunits, demonstrated that

Spodoptera frugiperda Sf 21 cells infected with either recombinant virus secreted mature homodimeric activin-A intothe medium. In addition, Sf 21 cells infected with the recombinant AcBovINHA virus were found also to producesubstantial amounts of the a-inhibin precursor protein. However, the mature portion of the latter is not secretedinto the medium but is retained within infected cells in an incompletely processed form(s). The recombinant activin-

A secreted by Sf 21 cells infected with the AcBovACTA virus was shown to possess activin bioactivity whenanalysed by in vitro bioassay and, therefore, provides an alternative route to mammalian cell expression for theproduction of recombinant activin-A. # 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Activin; Inhibin; Baculovirus; Recombinant expression; Spodoptera frugiperda Sf 21

1. Introduction

Activins and inhibins are dimeric peptide

hormones that, through their respective opposing

positive and negative in¯uences, contribute to

follicle-stimulating hormone (FSH) homeostasis,

thus providing an indirect control on the process

of folliculogenesis. In turn, FSH stimulates inhi-

The International Journal of Biochemistry & Cell Biology 30 (1998) 1129±1145

1357-2725/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S1357-2725(98 )00077-6

TheInternationalJournal of

Biochemistry& Cell Biology

PERGAMON

1 Present address: Molecular Biology Division, Veteran's

A�airs Medical Center (151-S), 4150 Clement Street, San

Francisco, CA 94121, USA.2 Present address: Department of Surgery, MSLS Building,

Room P228, Stanford University Medical Center, Stanford,

CA 94305-5486, USA.

* Corresponding author. Tel.: +353-1-706-2808; Fax:

+353-1-269-2016; E-mail: ®[email protected].

bin production in the granulosa cells of theovary, thereby modulating its own synthesis bya feedback mechanism (for a review see [1]).Biologically-active inhibin is comprised of an 18kDa mature a-subunit in disulphide-linkage witheither of two similar forms of a 14 kDa matureb-subunit, bA or bB, termed inhibin-A (abA) andinhibin-B (abB), whereas activins are formed bythe dimerisation of the mature b-subunits to pro-duce activin-A (bAbA), activin-B (bBbB) andactivin-AB (bAbB) (for reviews see [2, 3]). cDNAcloning of activin/inhibin mRNAs from a varietyof species has shown that all subunits are syn-thesised as preproprotein precursors. Sequencesimilarities and structural features have placedthe inhibin/activin subunits within the TGF-bsuperfamily of proteins (for reviews see [4, 5]). Inaddition to its role in folliculogenesis, activin-Ais identical with erythroid di�erentiation factor [6]and has recently been implicated in the processof embryogenesis (for a review see [7]) and tocause apoptosis in certain cell types [8].

To date, the majority of studies requiring asource of bioactive activin and/or inhibin havebeen carried out with preparations isolated frombovine or porcine follicular ¯uid or have utilisedrecombinant formulations of the human activin-A and inhibin-A molecules. The latter areproduced in cultured monkey kidney CV-1cells [9, 10] and limited amounts have beenmade available to researchers upon request toGenentech. In order to develop an alternativerecombinant source of these proteins and, in par-ticular, with the view to producing recombinantbovine inhibin with which to augment our bovinefecundity vaccine program, we chose to examinethe potential of the baculovirus expression vectorsystem as a means to produce recombinantactivin-A and inhibin-A. The isolation andcharacterisation of all three bovine inhibin/activin genes has been reported by thislaboratory [11]. The complexity of the intra- andintersubunit disulphide linkages in the inhibinand activin molecules (see e.g. [12]), togetherwith the essential roles of the pre- and proproteinsegments in the production of the mature dimers(see e.g. [13]) combine to exclude the applicationof a bacterial expression system to produce

functional recombinant activin or inhibin.Recombinant baculovirus-infected Spodopterafrugiperda cells have been used extensively inrecent years as a successful means to produce avariety of intracellular, membrane-associated andsecreted recombinant proteins requiring variousdegrees of post-translational processing for func-tional integrity (for a review see [14]). Recently,HuÈ sken-Hindi et al. [15] have described the ex-pression of a monomeric mutant form of rat acti-vin-A in Trichoplusia ni cells infected with arecombinant baculovirus.

The present report describes the constructionand testing of two recombinant baculoviruses,AcBovACTA and AcBovINHA, that weredesigned to elicit the secretion of recombinantbovine activin-A and inhibin-A, respectively,when introduced into Spodoptera frugiperda(Sf 21) insect cells. The results demonstrate thatinfection of Sf 21 cells with either virus results inthe secretion of mature functional homodimericactivin-A. However, although the bovine a-inhi-bin precursor protein is synthesised by Sf 21cells infected with the AcBovINHA virus, inhi-bin-A is not secreted by such cells but is retainedinternally in an incompletely processed form(s).

2. Materials and methods

2.1. Materials

5-Bromo-4-chloroindolyl phosphate and nitro-blue tetrazolium were purchased from Sigma.Oligonucleotide primers were synthesised in-house by using an Applied Biosystems 381ADNA Synthesiser.

2.2. Cells and viruses

Spodoptera frugiperda cells (IPLB-Sf-21; Sf 21cells) [16], virus stock of the BacPAK6 derivativeof the Autographa californica multiple nucleo-capsid polyhedrosis virus (AcMNPV) C6 andBsu36 I-digested BacPAK6 DNA were obtainedfrom Clontech Laboratories. All procedures per-taining to the maintenance of Sf 21 cells, thepropagation of recombinant viruses in Sf 21 cells,

C.N. Cronin et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1129±11451130

the titering of virus stocks and the preparation oflarge-scale amounts of virus stocks (Passage 2virus stock) were carried out as described in theBacPAK6 Laboratory Manual (Clontech) (seealso [17]) but with the exception that the volumeof viral inocula added to Sf 21 cell monolayers in35 mm Petri dishes was increased from 100 ml toa minimum of 170 ml to prevent substantial celldeath, through dehydration, in the centre of thedishes. Sf 21 cells were propagated routinely at278C in Grace's supplemented insect cell medium(GIMS; Gibco-BRL) containing 10% (v/v) foetalcalf serum (FCS) and in the presence of 50 unitsmlÿ1 penicillin and 50 mg mlÿ1 streptomycin(combined antibiotics stock solution from Gibco-BRL).

2.3. Construction of transfer vectors pAcBovACTA

and pAcBovINHA

A contiguous DNA sequence encoding thebovine a-inhibin preproprotein was assembled inpBluescript KS+ (Stratagene) by fusing a partial3 0-cDNA clone to a genomic fragment containingthe missing 5 0-segment as follows: a 907 bp Pst Ibovine genomic fragment surrounding the trans-lation start codon of the a-inhibin structural geneand cloned into the Pst I site of pBluescriptKS+ (a-cDNA-PI/KS+) [11] was digested withNco I/Xba I, blunt-ended with Klenow enzymeand re-circularised to generate pa5 0/KS+. A902 bp bovine a-inhibin partial cDNA (providedby Drs S. Sunderland and D.R. Headon; bases280±1182 of the sequence reported in [18]) thathad been cloned into the EcoR I site ofpBluescript KS+ (a-cDNA-EI/KS+) wasdigested to completion with BamH I/Hind III,followed by a partial digest with Pst I. Theresulting Pst I/Hind III fragment was ligatedto Pst I/Hind III-digested pa5 0/KS+ vector togenerate pa/KS+ containing a complete andcontiguous copy of the a-inhibin structural gene.The a-inhibin structural gene was transferred toM13mp18 by digesting pa/KS+ with Nde I/NotI, `blunt-ending' the released fragment withKlenow enzyme and cloning the latter into theHinc II site of the bacteriophage (Not I end of theinsert nearest the Hind III end of the M13mp18

polylinker) to generate pa/mp18. Attempts bysite-directed mutagenesis to introduce restrictionenzyme cloning sites adjacent to the start site oftranslation of the a-inhibin gene for cloning intothe baculovirus transfer vectors were unsuccess-ful. As described below, an alternative strategy,using PCR, was adopted to introduce these sites.

The 5 0-end of the a-inhibin structural gene wasmodi®ed for expression as follows: oligonucleo-tides oa5 [5 0-GGC AGG GCA GAT CTA AATATG TGG CTT CAG CTG CTC C-3 0] and`pUC/M13 reverse primer' (Promega) were usedin a polymerase chain reaction (PCR) to amplifya 320 bp fragment from pa5 0/KS+, followed by`blunt-ending' with T4 DNA polymerase [19] anddigestion with Sph I/Hind III. The 180 bp `blunt-end'/Sph I fragment was cloned into the pa/mp18vector, after digestion of the latter with Hind III,`blunt-ending' with Klenow enzyme and digestingsubsequently with Sph I. The resulting construct,pam/mp18, contained a contiguous copy of thea-inhibin structural gene with the desired altera-tions at the 5 0 end. DNA sequence analysis con-®rmed the integrity of the PCR process. Thecloning procedures described above are shownschematically in Fig. 1.

A contiguous DNA sequence encoding thebovine bA-inhibin/activin preproprotein ¯ankedby Bgl II restriction enzyme sites was assembledin pBluescript KS+ by fusing two PCR-ampli-®ed genomic DNA fragments as follows: oligonu-cleotides obA5 [5 0-C ACA ACA ACT CTA GATCTA AAT ATG CCC TTG CTC TGG CTGAGA GG-3 0] and obA5i [5 0-GGG CAG GCCAGT GCC TGA TTC CGC GAA CGT GATGAT CTC C-3 0], were used to amplify a 390 bpfragment comprising the 5 0-portion of the bovinebA-inhibin/activin structural gene from a 15 kbbovine genomic Xho I DNA fragment [11],followed by digestion with Xba I/AlwN I.Oligonucleotides, obA3i [5 0-T TCA TCT TCAGGC ACT GCC AGG AAG ACG CTG CAC-3 0] and obA3 [5 0-CCC AGA ATT CGA GATCTA TGA GCA ACC ACA CTC CTC C-3 0],were used in a separate PCR reaction to amplifyan 890 bp DNA fragment containing the 3 0-portion of the bA-inhibin/activin structural genefrom a 1.8 kb bovine genomic Xba I DNA

C.N. Cronin et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 1129±1145 1131

Fig. 1. Cloning strategy used to construct a contiguous copy of the bovine a-inhibin structural gene with modi®cations. Two bovine

a-inhibin partial cDNA clones, a-cDNA-PI/KS+ and a-cDNA-EI/KS+, that, between them, contained the entire a-inhibin cod-

ing sequence were fused together as outlined in the ®gure and as described in detail in Section 2. Subsequently, a Bgl II restriction

site and the sequence `AAAT' were introduced adjacent to the ATG translational start codon by using PCR.


fragment [11], followed by digestion with EcoRI/AlwN I. The Xba I/AlwN I and AlwN I/EcoR Ifragments were ligated simultaneously to XbaI/EcoR I-digested pBluescript KS+ to yield pbA/KS+ containing a complete and contiguous copyof the bovine bA-inhibin/activin structural gene.This procedure took advantage of the degeneracyof the genetic code and of the recognitionsequence for the restriction enzyme AlwN I(CAGNNN/CTG) to eliminate the single intronand to allow fusion of the two exons (the pro-cedure incorporated a silent mutation within thecodon for Thr131). The cloning procedures areoutlined schematically in Fig. 2. DNA sequenceanalysis of the ampli®ed fragments identi®eda number of Taq DNA polymerase-introducedmutations in the 3 0-exon that were corrected byreplacement with a restriction fragment from thewild type DNA.

A baculovirus transfer vector, pAcBovINHA,containing the bovine a-inhibin and bA-inhibin/activin structural genes under control of oppos-ing viral polyhedrin and p10 promotors, respect-ively, was constructed by transferring the a-inhibin structural gene from pam/mp18 as a BglII/BamH I fragment into the BamH I site ofpAcUW31 to yield pAcBova. Subsequently, thebA-inhibin/activin structural gene was transferredfrom pbA/KS+ as a Bgl II fragment intothe unique Bgl II site of pAcBova to yieldpAcBovINHA.

A baculovirus transfer vector, pAcBovACTA,containing the bovine bA-inhibin/activin struc-tural gene sequence under control of the viralpolyhedrin promotor, was constructed by clon-ing, in the desired orientation, the Bgl II frag-ment containing the bA-inhibin/activin structuralgene into the BamH I site of the baculovirustransfer vector pBacPAK1 (Clontech).

2.4. Generation of recombinant baculovirusesAcBovACTA and AcBovINHA

The recombinant baculoviruses, AcBovACTA

and AcBovINHA, were generated as follows: 0.5mg pAcBovACTA or pAcBovINHA plasmidDNA and 2 ml Bsu36 I-digested BacPAK6 viralDNA solution (Clontech) were diluted to 50 ml

with sterile water and mixed with 50 ml 0.1 mgmlÿ1 aqueous Lipofectin1 (Gibco-BRL). After15 min at room temperature, the mixture wasused to transfect Sf 21 cells as described in theBacPAK6 Laboratory Manual (Clontech) (seealso [17]). After 4 days the transfected cell super-natants were harvested and stored at 48C. Serialdilutions of the viral supernatants were used toinoculate Sf 21 cells in a plaque assay. Individualplaques were examined for the expression of theinhibin/activin subunits by Western blotting, asdescribed below.

2.5. Polyacrylamide gel electrophoresis andWestern blotting

Polyacrylamide gel electrophoresis in the pre-sence of sodium dodecyl sulphate (SDS-PAGE)was carried out using 13.75% (w/v) acrylamide/bisacrylamide resolving gels [19]. Reducing andnon-reducing conditions were in the presence andabsence, respectively, of 100 mM dithiothreitol(DTT) in the sample bu�er. Small-scale viral/Sf 21 culture supernatants were concentratedby mixing with an equal volume of 20% (w/v)trichloroacetic acid, placing at 48C overnight,centrifuging for 5 min and washing the pellettwice with 1 ml of acetone prior to suspension inSDS-PAGE sample bu�er. Cells were washedtwice, by centrifugation and resuspension, with1 ml of phosphate-bu�ered saline (PBS) beforesuspension in SDS-PAGE sample bu�er. Proteinsamples were placed in a boiling water-bath for5 min to aid dissolution although, in someinstances as indicated, this step was omitted toprevent b-elimination of intersubunit cystineresidues [20].

Fractionated proteins were transferred tonitrocellulose membranes (Schleicher and Schuell,Type BA 85) by using a semi-dry electroblotter(ATTO) at 0.65 mA cm2 for 2 h. The lane con-taining the protein standards was dissected awayand stained for protein by immersion for 2 minin a 0.1% (w/v) amido black solution prepared in45% (v/v) methanol/10% (v/v) acetic acid, fol-lowed by storage in a small volume of water. Theremainder of the ®lters were blocked by using1% (w/v) bovine serum albumin (Sigma A7906)


Fig. 2. Cloning strategy used to construct a contiguous copy of the bovine bA-inhibin/activin structural gene with modi®cations.

The two exons encoding the complete bA-inhibin/activin preproprotein were fused together by using a PCR strategy as outlined in

the ®gure and as described in detail in Section 2. During this process, Bgl II restriction sites, in addition to other cloning sites,

were introduced at either end of the bA-inhibin/activin structural gene, and also the sequence `AAAT' adjacent to the ATG transla-

tional start codon. The PCR primer sequences are shown in italics with non-matching (mutagenic) bases in bold type.


in PBS and probed with either monoclonal anti-body R1 [21] (Serotec, UK) or E4 [22] (Serotec,UK) at 1:2,000 dilution, speci®c for the matureportions of the a-inhibin and bA-inhibin/activinsubunits, respectively. The ®lters were probedsubsequently with a goat anti-mouse IgG (wholemolecule) secondary antibody conjugated toalkaline phosphatase (Sigma) at 1:10,000 dilutionand developed with 5-bromo-4-chloroindolylphosphate and nitroblue tetrazolium.

The migration distances of the protein stan-dards in each experiment were used to constructa semilogarithmic plot of log molecular massversus Rf . The relative molecular mass (Mr) ofthe immunoreactive activin and inhibin productswere estimated from data ®ts of the above valuesto third degree polynomials.

2.6. Protein expression studies

Protein expression studies with baculovirus-infected Sf 21 cells were carried out at 278C inserum-free Sf900 insect cell medium (Gibco-BRL) and in the presence of antibiotics (seeabove). For small-scale protein expression studiesin 35 mm Petri dishes, Sf 21 cells that had beenpropagated in GIMS containing 10% (v/v) FCSand antibiotics were used. Following viral inocu-lation, the infected monolayers were washedtwice with 1 ml Sf900 medium and incubated in2 ml Sf900 medium for the times indicated inthe ®gure legends. Intermediate-scale proteinexpression studies were conducted in 75 cm2

tissue culture ¯asks and were carried out byusing cells that had been adapted to growth inSf 900 medium as described in the BacPAK6 lab-oratory manual (Clontech). After culturing for 2days, the medium from infected cells was centri-fuged at 2,000g for 5 min to remove cells and thesupernatant concentrated by using a YM-3 mem-brane (Amicon). With the exception of the initialisolates, all protein expression studies utilised amultiplicity of infection of 10.

2.7. DNA sequencing

Single-stranded DNA templates of fragmentscloned into M13mp18 and/or M13mp19 were

sequenced by using the dideoxynucleotidechain termination method with [a-35S]dATP(Amersham) as label and by using T7 DNA poly-merase (Pharmacia). Compressions and/or cross-banding patterns in the sequencing ladders wereminimised, respectively, by using a deazaG com-mercial sequencing kit (USB) and by chasing thereactions with terminal deoxynucleotidyltransfer-ase (Promega) (see [23]).

2.8. In vitro activin bioassay

The functional integrity of activin-A may beassessed by determining its ability to stimulatedi�erentiation of cultured K562 erythroleukemiacells. This activity may be conveniently moni-tored during in vitro bioassay by measuring theaccumulation of haemoglobin in cultures ofK562 cells incubated with the hormone accordingto the procedures described by Schwall andLai [24]. Protein concentrations were determinedaccording to Bradford [25] and by using hen eggwhite lysozyme as protein standard.

3. Results and discussion

3.1. Construction of baculovirus transfer vectorspAcBovACTA and pAcBovINHA

The structures of the recombinant baculo-virus transfer vectors pAcBovACTA andpAcBovINHA are shown diagrammatically inFig. 3. pAcBovACTA contains a single contigu-ous copy of the bovine bA-inhibin/activin struc-tural gene sequence cloned into the uniqueBamH I site of the baculovirus protein expressiontransfer vector pBacPAK1 and under control ofthe viral polyhedrin promotor. pAcBovINHA

contains a single contiguous copy of each of thebovine a-inhibin and bA-inhibin/activin structuralgene sequences cloned into the unique BamH Iand Bgl II sites, respectively, of the baculovirusdual expression transfer vector pAcUW31 [26]and under control of viral polyhedrin and p10promotors, respectively. The tailoring proceduresused to transfer the a- and bA-inhibin/activingenes to the baculovirus transfer vectors,


described in Section 2, included the placement of

the sequence AAAT immediately preceding the

ATG translational start codons. It was con-

sidered that this manipulation might enhance the

production levels of the bovine inhibin/activin

proteins since this sequence has been found to

precede a number of highly expressed baculoviral

proteins [27].

Fig. 3. Schematic diagrams of baculovirus transfer vectors pAcBovACTA and pAcBovINHA. pAcBovINHA: a contiguous DNA

fragment encoding the bovine a-inhibin preproprotein was cloned into the unique BamH I site of the baculovirus dual expression

transfer vector pAcUW31 under control of the polyhedrin promotor. Into the unique Bgl II site of the resulting construct was

inserted a contiguous DNA fragment encoding the bovine bA-inhibin/activin preproprotein under control of the viral p10 promo-

tor. pAcBovACTA: a Bgl II fragment encoding the bovine bA-inhibin/activin preproprotein was cloned into the unique BamH I site

of the baculovirus transfer vector pBacPAK1 under control of the polyhedrin promotor. In each map the non-shaded box portions

represent viral sequences required for homologous recombination in vivo. The viral promotors are represented by black arrowheads

and the shaded areas downstream of the promotors represent the pre-, pro- and mature protein segments, in that order, of each

inhibin/activin subunit. The remaining sequences are required for maintenance and replication in E. coli. The sizes of the constitu-

ents within each diagram accurately re¯ect relative sequence lengths.


3.2. Generation of recombinant baculovirusesAcBovACTA and AcBovINHA

The recombinant baculoviruses AcBovACTA

and AcBovINHA were produced by co-transfection of Sf 21 cells with pAcBovACTA orpAcBovINHA, respectively, and Bsu36 I-digestedBacPAK6 viral DNA, as described in Section 2.In each case, three of the resulting viral plaqueswere examined for the production of thebovine bA-inhibin/activin subunit by Westernblotting. One plaque only in each group wasfound to express the bA-subunit (data notshown). These recombinant viruses were namedAcBovACTA and AcBovINHA as appropriate.The AcBovINHA virus was found also to expressthe a-inhibin subunit (see below). Preparationsof large-scale amounts of recombinant viruses(Passage 2 virus stocks) reached a titre of13�107 pfu mlÿ1.

3.3. Production time-course of the bovine bA-inhibin/activin subunit protein in Sf 21 cellsinfected with AcBovACTA

The time course of production of thebA-inhibin/activin subunit in Sf 21 cells infectedwith AcBovACTA was studied by Western blotanalysis using monoclonal antibody E4 [22] thatis speci®c for the mature portion of the bA-inhi-bin/activin preproprotein. At various times fromzero to 96 h post-infection (p.i.) individualcultures were separated into cellular and super-natant (medium) fractions and subjected toSDS-PAGE. As shown in Fig. 4, the bovinebA-inhibin/activin subunit is produced maximallyby day 3 of the infection cycle, consistent withthe known time-course of recombinant proteinexpression from the polyhedrin promotor (seee.g. [28]). Under non-reducing conditions(Fig. 4A), a major immunoreactive protein bandwith a Mr of 25,600 is present in the medium, inaddition to a number of minor protein bands ofwhich the most prominent migrate with Mr

values of 91,600, 59,400 and 54,900. Upon re-duction with DTT (Fig. 4B), all of the secretedimmunoreactive material resolves into three pro-tein bands with Mr values of 51,400, 15,300 and

13,900. Lesser amounts of the non-reduced91,600 and 25,600 Mr proteins are found alsoto be associated with the cellular fraction,suggesting that portions of these particular pro-tein species remain within the cells or are tightlyassociated with the cell surface.

The detection of the secreted 25,600 M r speciesthat resolves into sub-species of Mr 15,300and/or 13,900 is consistent with that expected forthe activin-A homodimer. The resolution of theactivin-A `homodimer' into two dissimilar-sizedprotein species following reduction has beenobserved previously with some other activinpreparations, including that isolated from porcinefollicular ¯uid [29] and the activin-A producedby mammalian cells infected with a recombinantvaccinia virus [30]. By way of explanation,the seeming incongruity that both species are de-rived from the activin-A `homodimer' may beaccounted for by considering the case whereby afraction of the mature bA-inhibin/activin subunitswithin the dimer population has undergoneinappropriate cleavage during maturation at adibasic site within the mature sequence. Themature bA-inhibin/activin subunit encompassesresidues 310±425 within the preproproteinsequence and follows a string of ®ve arginineresidues that, presumably, serves as the primaryrecognition sequence for proteolytic cleavageby the processing enzyme. However, residues322±323 and 411±412 within the mature subunitare each lysine [11]. Cleavage at the carboxyl sideat either of these sites would separate a fragmentof 1,508 Da (amino-terminal peptide) or 1,439Da (carboxy-terminal peptide) from the maturesubunit. However, within these fragments arecontained three and two cysteine residues respect-ively that, based on the work of Mason [12]and by analogy with the crystal structure ofTGF-b2 (cf. [12]), would be expected to allowthe bA-inhibin/activin dimer to retain all ofits constituent parts through disulphide bondinteractions. Upon reduction two major bandswould be generated di�ering in molecular massby approximately 1,500 Da, precisely the di�er-ence observed experimentally (Fig. 4B). Such anexplanation is consistent also with the resultsof site-directed mutagenesis studies indicating


that the cysteine residue at position 389 inthe bA-inhibin/activin sequence solely is respon-sible for mature subunit dimerisation throughthe formation of an intersubunit disulphidebond [12, 15].

The materials of greatest relative molecularmass detected in non-reduced samples from themedium of infected cells (Fig. 4A) likely rep-resent various partly processed forms of the acti-vin-A molecule, such as the proprotein

homodimer (theoretically 90,270 Da) and theproprotein-mature protein heterodimer (theoreti-cally 58,102 Da). Upon reduction these formsresolve into the proprotein species (theoretically45,135 Da) and the mature subunit. This diver-sity may be more elaborate depending on thestate(s) of glycation of the pro region of thevarious protein species. A similar mixture ofpartly processed forms has been observed in themedium of mammalian cells expressing recombi-

Fig. 4. Time-course of expression of the bovine bA-inhibin/activin subunit in Sf21 cells infected with the recombinant baculovirus

AcBovACTA. At the times indicated, individual cultures of Sf21 insect cells that had been infected with AcBovACTA were separ-

ated into cell (c) and supernatant (s) fractions and samples of each were subjected to SDS-PAGE/Western analysis. The leftmost

lane of each blot contains a set of protein molecular mass standards that was dissected away prior to immunodetection and stained

for protein as described in Section 2. The remainder of the membranes were probed with monoclonal antibody E4 that is speci®c

for the mature portion of the bA-inhibin/activin subunit. (A) The samples were not reduced prior to electrophoresis. (B) The

samples were treated with 100 mM DTT prior to electrophoresis.


nant human activin-A [10, 31]. By analogywith these other expression systems (e.g. [10]) itis likely that the released pro region alone issecreted also by Sf 21 cells infected with theAcBovACTA virus but is not observed in the pre-sent study because the monoclonal antibodiesused in the detection system are directedspeci®cally towards the mature portion of thebA-inhibin/activin protein.

3.4. Production time-courses of the bovine a- andbA-inhibin/activin subunit proteins in Sf 21 cellsinfected with AcBovINHA

The time courses of production of the a- andbA-inhibin/activin subunits in Sf 21 cells infectedwith AcBovINHA were studied also by Westernblot analysis using monoclonal antibodies R1 [21]and E4 that are speci®c for the mature portionsof the a- and bA-inhibin/activin preproproteins,respectively. At various times from zero to 96 hp.i. individual cultures were separated into cellu-lar and supernatant (medium) protein fractionsand subjected to SDS-PAGE. As shown in Fig. 5,the time-courses of production of the bovine a-and bA-inhibin/activin subunits are consistent,respectively, with the known time-courses ofrecombinant protein expression from the poly-hedrin and p10 promotors (see e.g. [28]), withmaximal accumulation of product by day three.However, as Fig. 5 shows, the material that isimmunoreactive with the anti-a-inhibin maturesubunit monoclonal antibody R1 occurs onlyin the cellular fraction. Under non-reducingconditions (Fig. 5A), this material is of highmolecular mass with a smeared appearance,although two protein bands are discernible withMr values of 48,500 and 44,700. In the presenceof DTT (Fig. 5B), the appearance of theimmunoreactive material in the cellular fractionis no longer smeared but resolves into threeprominent protein species with Mr values of48,400, 46,700 and 43,100. These forms likelyrepresent the a-inhibin proprotein with varyingextents of glycation. As many as six di�erentinhibin forms ranging in Mr from 32,000 to120,000 have been identi®ed in bovine follicular

¯uid [32]. The possibility exists that glycationevents in the insect cells might mask the antigeni-city of any mature recombinant inhibin towardsthe R1 monoclonal antibodies. However, this isan unlikely event since glycation is known to beless complex in insect cells, yet the R1 antibodydoes recognise fully glycated mature mammalianinhibin. In addition, it would be expected thatthe anti-activin E4 monoclonal antibody wouldreact with any such species, if present, in theinhibin producing cells, which, as describedbelow, is not the case.

In contrast to the a-inhibin subunit, much ofthe bA-inhibin/activin subunit expressed in Sf 21cells infected with the AcBovINHA virus issecreted into the medium and, as shown inFig. 5C, the various forms secreted mirror thoseproduced by Sf 21 cells infected with theAcBovACTA virus. However, a comparison ofFig. 4A and Fig. 5C indicates that, unlike thecase with AcBovACTA, a substantial fraction ofthe expressed bA-inhibin/activin subunit proteinremains associated with those cells infectedwith the AcBovINHA virus. This suggests thatthe expressed a-inhibin protein interacts insome way with this fraction of the expressed bA-inhibin/activin protein, preventing secretion ofthe latter. Thus, it seems likely that some form ofdimerisation of the a- and bA-subunits doesoccur in AcBovINHA-infected cells. This dimeri-sation is most likely to occur within theendoplasmic reticulum, and suggests that the pre-sequence of the a-inhibin subunit is active indirecting the nascent a-inhibin preproprotein intothat compartment. However, the ®nding thatthe mature inhibin-A dimer is absent not onlyfrom the extracellular medium but also fromthe cell fraction suggests that the proteolyticprocessing site that precedes the mature a-inhibinsubunit is either not recognised or is ine�cientlyprocessed in Sf 21 cells, a situation that preventssecretion. Alternatively, since it is well knownthat Spodoptera frugiperda insect cells do notgenerally glycate proteins to the same degreeof complexity as that observed in mam-malian cells (see [14]) it may be that the maturea-inhibin subunit requires a more elaborate ad-dition of oligosaccharides for proper maturation


Fig. 5. Time-courses of expression of the bovine a- and bA-inhibin/activin subunits in Sf21 cells infected with the recombinant

baculovirus AcBovINHA. At the times indicated in the ®gures, individual cultures of Sf21 insect cells that had been infected with

AcBovINHA were separated into cell (c) and supernatant (s) fractions and samples of each were subjected to SDS-PAGE/Western

analysis. The leftmost lane of each blot contains a set of protein standards that was treated as described in the legend to Fig. 4.

(A) The membrane was probed with monoclonal antibody R1 that is speci®c for the mature portion of the a-inhibin subunit. The

samples were not reduced prior to electrophoresis. (B) As in A except the samples were treated with 100 mM DTT prior to electro-

phoresis. (C) The membrane was probed with monoclonal antibody E4 that is speci®c for the mature portion of the bA-inhibin/activin subunit. The samples were not reduced prior to electrophoresis.


and subsequent secretion of the inhibin dimer(unlike the mature activin molecule, maturenative inhibin is known to be glycated; seee.g. [29]).

3.5. Analyses of a- and bA-inhibin/activin subunitproteins produced by intermediate-scale cultures ofSf 21 cells infected with AcBovACTA andAcBovINHA

Observations made by light microscopy(data not shown) indicated that Sf 21 cellsinfected with either recombinant baculovirus,AcBovACTA or AcBovINHA, exhibited a morerapid onset of cell lysis than did cells infectedwith the parental BacPAK6 baculovirus. Celllysis was not observed with mock-infected cells.A report by Nishihara et al. [8] describing theapoptotic e�ects of activin on a number ofmammalian cell lines suggests that the obser-vations made in the present study might re¯ect asimilar action of recombinant bovine activin-Aon Sf 21 insect cells. In light of this, intermedi-ate-scale harvests of material secreted into themedium by Sf 21 cells grown in 75 cm2 tissue cul-ture ¯asks and infected with either AcBovACTA

or AcBovINHA were conducted after 2 days ofviral infection, in order to minimise contami-nation with cellular material. Mock-infected cellsand cells infected with the parental BacPAK6virus were employed as controls.

The concentrated media from mock-,BacPAK6-, AcBovACTA- and AcBovINHA-infected Sf 21 cell cultures were analysed forthe presence of activin-A and inhibin-A sub-units by Western blotting as described above.Proportional amounts of the cellular fractionswere electrophoresed side by side with the extra-cellular material to allow for direct comparisons.The data in Fig. 6 indicate that only those cul-tures infected with AcBovACTA or AcBovINHA

produce signi®cant amounts of the bA-inhibin/activin subunit whereas only AcBovINHA-infected cultures produce signi®cant amounts ofthe a-inhibin subunit. A weak cross-reactionof both monoclonal antibodies against theb-galactosidase protein (116 kDa) that isproduced in the BacPAK6-infected cells is

apparent. As found with the small-scale cul-tures described above, a comparison of theAcBovACTA- and AcBovINHA-infected culturesindicates that although both produce similaramounts of the bA-inhibin/activin subunit, themajority produced by the AcBovACTA-infectedcells is secreted as the mature activin-A homo-dimer but that a signi®cant proportion of thebA-inhibin/activin subunit produced by theAcBovINHA-infected cells remains associatedwith the cellular fraction in an incompletelyprocessed form(s).

3.6. In vitro bioassay of recombinant bovineactivin-A

The concentrated media from intermediate-scale cultures of Sf 21 cells infected with eitherBacPAK6 or AcBovACTA (see above) weretested for activin bioactivity as described inSection 2. As shown in Fig. 7, only the concen-trated medium from insect cell cultures infectedwith the AcBovACTA virus was capable of sti-mulating the di�erentiation of K562 human ery-throleukemia cells, monitored by measuringhaemoglobin accumulation [24], indicating thepresence of bioactive activin-A. The decrease inthe accumulation of haemoglobin at the highestconcentration of extract may re¯ect some deleter-ious e�ect(s) of high doses of the hormone orsome other component of the crude extract orcell-free medium on cell viability. A similaramount of protein from the concentrated med-ium of Sf 21 cells infected with the BacPAK6virus was without e�ect on haemoglobin accumu-lation (Fig. 7). The amount of crude activin-Aprotein required to produce a half-maximal re-sponse was 1.3120.03 mg mlÿ1. SDS-PAGEanalysis of this crude material identi®ed a varietyof proteins, indicating that the true speci®c ac-tivity of the recombinant material is greater thanthis value.

4. Conclusions

Two recombinant Autographa californica bacu-loviruses, AcBovACTA and AcBovINHA, were


constructed that were designed to elicit the se-cretion of recombinant bovine activin-A andinhibin-A, respectively, when introduced intoSf 21 insect cells. The results of Western blotanalyses indicate that Sf 21 cells infected withAcBovACTA secrete mature activin-A into the

medium, in addition to a variety of incompletelyprocessed forms that re¯ects those found tooccur naturally. The material secreted fromAcBovACTA-infected Sf 21 cells was shown topossess activin bioactivity. The baculovirus sys-tem thus provides an alternative route to cultured

Fig. 6. Analyses of bovine a- and bA-inhibin/activin subunit expression in intermediate-scale cultures of Sf21 cells either mock-

infected or infected with BacPAK6, AcBovACTA or AcBovINHA recombinant baculoviruses. Cultures of Sf21 cells in 75 cm2 tis-

sue culture ¯asks were either mock-infected or infected with BacPAK6, AcBovACTA or AcBovINHA baculoviruses at a multi-

plicity of infection of 10. After two days the cultures were separated into cell (c) and supernatant (s) fractions. Concentrated

sample volumes equivalent to 120 ml of the original supernatants and proportional amounts of the cellular material were subjected

to SDS-PAGE/Western analysis. The samples were neither reduced nor heated prior to electrophoresis. The leftmost lane of each

blot contains a set of protein standards that was treated as described in the legend to Fig. 4. (A) The membrane was probed with

the monoclonal antibody R1 that is speci®c for the mature portion of the a-inhibin subunit. (B) The membrane was probed with

monoclonal antibody E4 that is speci®c for the mature portion of the bA-inhibin/activin subunit.


mammalian cells for the production of functionalrecombinant activin. The identical amino acidsequences of the mature portions of the bA-subu-nit among all mammalian species examined todate, together with the absence of glycation ofthe activin molecule, indicates that the recombi-nant bovine activin-A produced in the presentstudy is likely equivalent to the human protein.Future experiments with this system will be di-rected towards scaling-up the process in order toisolate the recombinant hormone in pure formfor further study.

In the case of the AcBovINHA virus, theresults of Western blot analyses indicate that

Sf 21 cells infected with this virus produceboth the a- and the bA-inhibin/activin subunitsrequired for synthesis of the inhibin-A molecule.However, although dimerization does appearto occur between these two subunits, the resultsindicate that such complexes are incompletelyprocessed and, perhaps as a result, are notsecreted from infected cells. Future exper-iments will attempt to rectify this situation byreplacement of the a-inhibin pro- and/or pre-region with those from the bA-inhibin/activinprotein and/or by removal of the glycation site(s)within the a-inhibin sequence by site-directedmutagenesis.

Acknowledgements

We are grateful to Drs S. Sunderland(University College, Dublin) and D. R. Headon(University College, Galway) for providing thepartial cDNA of the bovine a-inhibin subunitand to Dr Tommy Gallagher (University College,Dublin) for oligonucleotide synthesis. We thankDr Nicola Gardiner (St. James Hospital, Dublin)for providing initial stocks of K562 cells.Funding for this project was provided by theNational Agricultural and VeterinaryBiotechnology Centre (NAVBC; a division ofBioResearch Ireland), University College Dublin.We are particularly grateful to Dr Michael Foxeof the NAVBC for his support of this project.

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