Engineering of a Pichia pastoris expression system for

Journal of Biotechnology116(2005)
251–260
Engineering of a Pichia pastoris expression system for
secretion of high amounts of intact human
parathyroid hormone
Randi Vad∗,Eidi Nafstad,Linn Anita Dahl,Odd S.Gabrielsen
Department of Molecular Biosciences,University of Oslo,P.O.Box1041Blindern,N-0316Oslo,Norway
Received20August2004;received in revised form19November2004;accepted6December2004
Abstract
Human parathyroid hormone(hPTH)is involved in calcium metabolism,and the unique ability of this hormone to stimulate bone growth makes it a promising agent in the treatment of osteoporosis.We have engineered the methylotrophic yeast Pichia pastoris for the production of over300mg intact hPTH per liter growth medium.The presence of10mM EDTA in the culture medium was essential to obtain this high hormone yield,indicating that metallopeptidases are mainly responsible for the otherwise instability of hPTH.Furthermore,the secretion process of hPTH was considerably improved by coexpression of Saccharomyces cerevisiae protein disulphide isomerase(Sc PDI).Since hPTH does not contain any cystein residues,this effect may be indirect or ascribed to the chaperone activity of PDI.Contrary to the situation in S.cerevisiae,use of a protease-deficient host strain provided no additional advantage.The hormone secreted by P.pastoris was not subjected to proteolytic processing by Kex2p in the two internal tribasic sites,nor were any C-terminal truncated hPTH forms detected.However,the P.pastoris hPTH producing transformants cosecreted ubiquitin to the culture medium,possibly as a result of a stress-related response.
©2004Elsevier B.V.All rights reserved.
Keywords:Pichia pastoris;Human parathyroid hormone;Heterologous protein expression;Protein disulphide isomerase;EDTA;Ubiquitin
Abbreviations:AOX1,alcohol oxidase gene;EDTA,ethylene diamine tetraacetic acid;EGTA,ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N ,N -tetraacetic acid;hPTH,human parathyroid hormone;PDI,protein disulphide isomerase;PpPDI and ScPDI,the coding region of the protein disulphide isomerase gene from P.pas-toris and S.cerevisiae,respectively
∗Corresponding author.Tel.:+4722856169;
fax:+4722854443.
E-mail address:randi.vad@biokjemi.uio.no(R.Vad).1.Introduction
Human parathyroid hormone(hPTH)is a peptide hormone of84amino acids that serves as the princi-pal regulator of calcium homeostasis and bone turnover in vertebrates by activating specific receptors located on osteoblastic and renal tubular cells(Potts et al., 1995).The unique ability of this hormone to increase bone density makes it a promising agent in the treat-
0168-1656/$–see front matter©2004Elsevier B.V.All rights reserved. doi:10.1016/j.jbiotec.2004.12.004
252R.Vad et al./Journal of Biotechnology116(2005)251–260
ment of osteoporosis(Neer et al.,2001;Whitfield et al.,2002;Rubin and Bilezikian,2003).The anabolic and classical cAMP-stimulating activities of PTH have primarily been assigned to the N-terminal portion, PTH(1–34),which is sufficient for complete activation of the G-protein-coupled parathyroid hormone1recep-tor(PTH1R).The1–14portion of PTH is a determi-nant of receptor activation for second-messenger sig-naling(Luck et al.,1999),and the15–34portion con-trolling receptor binding affinity(Caulfield et al.,1990; Bergwitz et al.,1996).The signal transduction mecha-nism of PTH1R has been described as a two-site model, in which the C-terminal part of the ligand interacts with the large extracellular N-terminal domain of the recep-tor,and the N-terminal region of the ligand interacts with the juxtamembrane domain comprising the trans-membrane␣-helices and interconnecting loops.This model has later been extended to take into account the effect of different receptor conformational states aris-ing from receptor-G-protein interaction on the molecu-lar mechanism of ligand binding to the PTH1receptor (Hoare et al.,2001;Chorev,2002).
Various lines of evidence suggest that the mid-and C-terminal regions of PTH have more physio-logical relevance than previously thought.The mid-region,PTH(28–48),had a strong in vivo anabolic effect on neonatal mouse bones,possibly in inter-play with IGF-I(Rihani-Bisharat et al.,1998).C-terminal PTH fragments stimulated gene expression of the two bone formation markers type-1procol-lagen and IGFBP-5in osteoblastic UMR-106cells (Nasu et al.,1998),while residues73–76were es-sential for activation of a nickel-insensitive Ca2+in-flux pathway in growth plate chondrocytes(Erdmann et al.,1998).Receptors that specifically recognize the C-terminal region of intact PTH and circulating C-terminal fragments were highly expressed in osteocytic cells,where they may play a role in regulating cell sur-vival and intercellular communication(Divieti et al., 2001).Furthermore,human PTH(7–84)influenced the Ca2+concentration negatively in thyroparathyroidec-tomized rats,thereby limiting the calcemic responses to hPTH(1–84)and hPTH(1–34),and inhibited bone resorption in vitro by acting on receptors distinct from PTH1R(Nguyen-Yamamoto et al.,2001;Divieti et al., 2002).
Elucidation of the biological activities of hPTH re-lated to the structure of the hormone is of consider-able medical importance,as more information con-cerning regulation of bone formation and character-ization of molecular interactions between the ligand and receptor should make it possible to create more potent therapeutic agonists.Such studies obviously re-quire access to large amounts of pure hormone,which can be achieved by heterologous production in yeast or Escherichia coli.The most effective bacterial expres-sion system reported produced a fusion protein,where hPTH was obtained as[Pro−1]-hPTH(1–84)after acid cleavage(Paulsen et al.,1995).Another strategy in-cluding expression of a synthetic hPTH gene under the control of the T7promoter in E.coli resulted in N-terminally correct hormone,but a lower hormone yield (Oshika et al.,1994).One of the advantages with yeast as host organism is the effective secretion apparatus, which facilitates the purification procedure of the for-eign protein.The combination of a growth medium rich in amino acids and a protease-deficient host strain was essential to obtain intact hPTH in Saccharomyces cere-visiae(Gabrielsen et al.,1990).The expression system was further improved by elimination of the spacer pep-tide in the MF␣fusion(Gabrielsen et al.,1990)and one of two internal potential Kex2p processing sites by K26Q mutation,which resulted in a more degradation-resistant hPTH agonist,hPTH Q(Reppe et al.,1991). Two biologically active O-glycosylated hPTH forms were identified among minor products(Olstad et al., 1992).Finally,the level of intact K26Q mutated hor-mone was enhanced30-fold to16mg l−1by changing from the constitutive MF␣promoter to the inducible CUP1promoter in the expression cassettes,use of an-other host strain,and optimization of fermentation con-ditions(Vad et al.,1998).
In the present work,we have explored the potential of the methylotrophic yeast Pichia pastoris to secrete intact hPTH.This yeast has the ability to express and se-crete foreign proteins at high levels,and perform com-plex post-translational modifications(Cereghino and Cregg,2000).Changing from the S.cerevisiae hPTH expression system to P.pastoris considerably increased the yield of intact non-mutated hormone,which was as stable as hPTH Q in this expression system.Coexpres-sion of PDI from S.cerevisiae(Sc PDI)in the hPTH expressing cells and addition of EDTA to afinal con-centration of10mM in the culture medium contributed to a maximum level of over300mg hPTH per liter growth medium.
R.Vad et al./Journal of Biotechnology116(2005)251–260253
2.Materials and methods
2.1.Host strains and vectors
The P.pastoris strains GS115(his4)and SMD1163 (his4,pep4,prb1),and the vector pHIL-D5(AOX1 promoter,HIS4,Kan R,Eco RI cloning site)were kindly provided by Phillips Petroleum Co.The vector pPICZ,B was from Invitrogen life tech.
2.2.Media composition
Culture media components were from Difco Lab-oratories.RDB His−medium contained1M sorbitol, 2%glucose,1.34%yeast nitrogen base(YNB),and 0.4␮g biotin and50mg amino acid mixture per ml−1. MD contained2%glucose,1.34%YNB and0.4␮g bi-otin per ml−1.MM contained0.5%methanol,1.34% YNB and0.4␮g biotin per ml−1.BMGY consisted of 1%glycerol,1%yeast extract,2%peptone,100mM potassium phosphate pH5.8,1.34%YNB and0.4␮g biotin per ml−1.BMMY was as BMGY with the ex-ception that1%methanol replaced the glycerol.YPD contained1%yeast extract,2%peptone and2%glu-cose.Solid media contained in addition2%agar or agarose,and the YPD plates100␮g ml−1zeocin(In-vitrogen life tech).
2.3.Construction of hPTH and ScPDI expression cassettes
A S.cerevisiae MF␣prepro sequence fused up-stream to cDNA encoding either hPTH or hPTH Q was isolated as a562bp Xho I–Xba I fragment from p␣UXPTH-2(Gabrielsen et al.,1990)and p␣UXPTH-Q26(Reppe et al.,1991),respectively,after insertion of a Xho I site downstream the MF␣-promoter in these plasmids(Reppe et al.,unpublished).The MF␣prepro-hPTH/hPTH Q cDNA fragments,lacking the spacer peptide codons,were inserted into thefilled-in unique Eco RI site between the AOX1promoter and terminator in pHIL-D5to generate the plasmids pHILD5-hPTH and pHILD5-hPTH Q.A1569bp DNA fragment con-taining the coding region of PDI andflanking Xba I restriction sites was amplified from S.cerevisiae by PCR.The PCR fragment was digested with Xba I,and subcloned in pUC19before transferring into the Xba I site of pPICZ,B.
2.4.Transformation of P.pastoris and screening
for multicopy transformants
The P.pastoris strains GS115and SMD1163were transformed by the spheroplast method with Not I frag-ments of pHILD5-hPTH and pHILD5-hPTH Q(Fig.1), and plated on RDB His−medium.Mut phenotypes were determined by streaking the colonies onto MD and MM agar plates,while multicopy transformants were screened for by dot–blot analyses(Romanos et al.,1991)and resistance level against4–10mg ml−1 geneticin(GibcoBRL).The transformants GS115/K10 and GS115/K23were retransformed with pPICZ,B-Sc PDI by the LiCl method.
2.5.Determination of copy number
Genomic yeast DNA from untransformed strains and selected transformants were isolated using conven-tional methods and purified on CsCl gradient.Kpn I-digests of total-DNA(3␮g)blotted onto Hybond N+filter(Amersham Biotech.)were hybridized with a ra-dioactive612bp Kpn I-fragment of the HIS4gene.
Ra-Fig.1.Schematic representation of the hPTH expression cassette in pHILD5-hPTH.The expression of hPTH cDNA is regulated by the methanol-inducible alcohol oxidase1(AOX1)promoter,and the translation product of the S.cerevisiae MF␣-factor leader sequence directs secretion into the culture medium.The histidinol dehydrogenase gene(HIS4)allows selection of recombinant clones,while the geneticin resistance gene (Kan R)facilitates selection of transformants having multiple copies of the expression cassette in the genome.The3 AOX1TT sequence is the native termination and polyadenylation signal of the AOX1gene.The hPTH Q expression cassette of pHILD5-hPTH Q,encoding the K26Q mutant,is identical to pHILD5-hPTH except for containing hPTH Q cDNA instead of hPTH cDNA.
254R.Vad et al./Journal of Biotechnology116(2005)251–260
dioactive spots were cut out from thefilter and cpm reg-istered by scintillation counting.The references were total-DNA from untransformed strains containing only one copy of His4.
2.6.Sequencing of PCR-amplified hPTH expression cassettes from the transformants The hPTH expression cassette in selected transfor-mants was amplified from total-DNA by PCR using the primer set5 -TTTCATAATTGCGACTGGTTCC-3 /5 -CTGCATCTCTCAGGCAAATGG-3 comple-mentary toflanking vector sequences.The products were purified and cycle sequencing reactions were set up with the Thermo Sequenase system(Amer-sham Bioscences)and Cy5-labelled primers starting inside the PCR primers.Automatic DNA-sequencing was performed by an ALFexpress DNA sequencer (Pharmacia Biotech.).
2.7.Culture conditions
Expression trials were performed by growing the transformants in10ml BMGY medium for2days,af-ter which the cells were sedimented by centrifugation and resuspended into2ml BMMY medium for another 48h.Methanol was added to afinal concentration of 1%every24h.The transformants secreting the highest level of hPTH were cultivated in shakeflasks with1l BMGY medium for2days before induction in100ml BMMY medium for24–144h.Standard growth tem-perature was30◦C.
2.8.Northern analyses
Isolation of total-RNA was performed according to standard procedures including breakage of the cell wall with glass beads.A479bp fragment of the AOX1gene (encompassing−100to+379),hPTH cDNA cut out from pUC19with Eco RI/Hin dIII,and a1569bp Xba I fragment of Sc PDI from pPICZ,B-Sc PDI were used as probes,respectively.
2.9.Purification and characterization of secreted hPTH
Cells were removed by centrifugation and yeast cul-ture media acidified to pH3with HCl prior to concen-tration by passage through a SP Sepharose Fast Flow Column(Pharmacia)essentially as described(Reppe et al.,1991;Olstad et al.,1992).Aliquots of the fractions containing the highest level of hPTH were subjected to analytical reverse-phase chromatography by an¨AKTA TM purifier10in two steps.First,the fractions were eluted with a linear gradient from20 to60%eluant B1(eluant A1:0.065%trifluoroacetic acid in2%aqueous acetonitrile,and eluant B1:0.05% trifluoroacetic acid in100%acetonitrile)on a Vydac protein/peptide C18column(4.6mm×250mm). Secondly,hPTH was reinjected onto a Sephasil protein C4column(4.6mm×250mm),which was isocratic eluted by46%B2(eluant A2:0.2%trifluoroacetic acid in MilliQ water,eluant B2:0.2%trifluoroacetic acid in70%aqueous acetonitrile).Mass spectrom-etry(MS)was accomplished on a MALDI-TOF V oyager TM-RP-DE Biospectrometry TM Workstation, while proteins to be sequenced were subjected to automated Edman degradation using an on-line pulsed phase sequenator(Applied Biosystems477A/ 120A).
2.10.Quantification of hPTH
Levels of intact hPTH secreted to the growth medium were estimated by comparing with a PTH stan-dard(from Bachem Inc.)after SDS–PAGE(15%)and silver staining,or quantified using OCTEIA Intact PTH Kit(Immuno Diagnostic Systems Ltd.).The hPTH pep-tides then react with a N-terminal specific monoclonal anti-hPTH(1–34)on the inner surface of polystyrene microtiter wells and secondary biotinylated affinity-purified goat anti-hPTH(39–84).
3.Results
3.1.Secretion of hPTH from P.pastoris
The P.pastoris strain GS115was transformed with the NotI-cut hPTH expression plasmid pHILD5-hPTH (Fig.1),and the His+transformants were screened by geneticin resistance level and dot–blot analyses for the presence of multiple copies of integrated hPTH ex-pression cassettes.The multicopy clones,containing at least15integrated copies of the expression cas-sette,secreted a markedly higher level of hPTH than
R.Vad et al./Journal of Biotechnology116(2005)251–260255 single-copy clones as estimated by SDS–PAGE.Two
of the highest expressing transformants with different
Mut phenotypes,GS115/K10(Mut+)and GS115/K23
(Mut s),were selected for further studies.The integrity
of hPTH cDNA in the integrated expression cassettes
of these transformants was confirmed by PCR amplifi-
cation and sequencing.
A maximum in hPTH mRNA level was reached72h
after induction(results not shown),while the highest
hormone yield was attained at an induction period of
about96h in BMMY medium,pH5.8.At these cul-
ture conditions,GS115/K10and GS115/K23secreted
6and33mg of intact hormone per liter,respectively
(Table1).Two prominent bands were though detected
by SDS–PAGE of culture media samples.In addition
to the protein band that corresponded to intact hPTH
in mobility,there was a protein band with an appar-
ent molecular mass of about6kDa that might consist
of one or more proteolytic products(Fig.2A).This
band,which appeared about24h after induction and
increased in time with intact hPTH,was not present
in control media of nontransformed cells.The follow-
ing experiments aimed to increase the yield of intact
hPTH,and to identify the secreted material in the6kDa
band.
Table1
Yield of secreted hPTH in the growth medium of P.pastoris trans-
formants cultivated at different culture conditions
Transformant Medium plus Induction
period(h)hPTH (mg l−1)
SMD1163/K146963
SMD1163/Q143963
GS115/K10966
GS115/K105mM EDTA9633
GS115/K1010mM EDTA9653
GS115/K23MM,1%Casaa727
GS115/K2310mM EDTA7235
GS115/K239633
GS115/K235mM EDTA9649
GS115/K2310mM EDTA9667
GS115/K2312028
GS115/K235mM EGTA12053
GS115/K235mM EDTA12055
GS115/K2310mM EDTA120127
GS115/K23-PDI10mM EDTA120349 Growth medium is BMMY if not specified,and the EDTA and EGTA concentrations in this medium are given.The values are the average of parallel cultures.The samples were quantified with Octeia Intact PTH
Kit.Fig.2.(A)Effect of EDTA in the growth medium.Silver stained gel (15%)after SDS–PAGE of30␮l culture medium from GS115/K23 induced for96h without EDTA(lane1)and in the presence of10mM EDTA(lane2),respectively.(B)Effect of EDTA in the growth medium combined with coexpression of Sc PDI in the hPTH produc-ing cells.1:GS115/K10induced for96h;2:GS115/K23induced for96h;3:GS115/K23induced for120h;4:GS115/K23induced for120h in the presence of10mM EDTA;5:GS115/K23-Sc PDI induced for120h in the presence of10mM EDTA.Values shown are the mean of parallel cultures.Error bars indicate S.D.
3.2.Approaches to increase the level of intact hPTH
Next,we asked whether the P.pastoris hPTH ex-pression system could be further improved by sim-ilar strategies as used for S.cerevisiae,namely use of a protease-deficient host strain combined with an enriched medium(Gabrielsen et al.,1990),use of constructs for production of the K26Q mutant hPTH Q form where a Kex2p sensitive site has been removed(Reppe et al.,1991),andfinally by opti-
256R.Vad et al./Journal of Biotechnology116(2005)251–260
mization of the growth conditions(Vad et al.,1998). Accordingly,the protease-deficient strain SMD1163 was transformed with the plasmids pHILD5-hPTH and pHILD5-hPTH Q(Fig.1).Two multicopy transfor-mants,SMD1163/K146(Mut+)and SMD1163/Q143 (Mut s),producing hPTH and hPTH Q respectively from approximately the same copy number,were selected. The band patterns on SDS–PAGE gels of secreted prod-ucts from the SMD1163transformants were identical to those from the GS115transformants,and there was no difference in the amount of intact hPTH and the agonist form hPTH Q(Table1).Hence,the yield of in-tact hPTH secreted from P.pastoris transformants in shakeflask cultures appears not to be influenced by the activity of proteinase A and B or carboxypeptidase Y,which should be eliminated in the SMD1163host. Neither is the tribasic site R25K26K27in hPTH sub-jected to Kex2p-like processing during secretion in P. pastoris,as is the case in the S.cerevisiae expression systems.
The most productive transformant,GS115/K23,was used to examine the influence of various culture condi-tions on the hormone level.The highest level of intact hPTH was obtained by induction in BMMY medium, probably due to a propitious pH value(5.8)along with a stabilizing effect of yeast extract and peptone. No intact hPTH was detectable in minimal medium (MM),where the pH increased to above7.5during the induction period.Supplementation with both1% casamino acids and phosphate buffer,pH5.8,to this medium was essential to get intact hormone,but at a low level(Table1).Lowering the temperature or pH during induction did not improve the yield of hPTH, which evidently is exposed to attack by extracellular proteases.
3.3.Increased yield of hPTH by addition of protease inhibitors
The instability of secreted heterologous proteins in the growth medium is perhaps the main problem with P.pastoris as host organism.With the intent to reduce the proteolysis of hPTH,we added differ-ent protease inhibitors to the BMMY medium at the time of induction of the GS115transformants.The in-hibitors were EDTA,EGTA,L-Arg,benzamidin and a complete TM Mini EDTA-free tablet(a mixture of pro-tease inhibitors that inhibit a broad spectrum of ser-ine and cysteine proteases)from Roche,respectively. Addition of EDTA to afinal concentration of5mM increased the level of intact hormone considerably,af-ter120h the GS115/K23transformant produced55mg intact hPTH per liter growth medium.When thefi-nal concentration of EDTA was doubled to10mM,the maximum level of intact hPTH secreted by GS115/K23 increased to over100mg l−1(Table1,Fig.2).Higher EDTA concentrations in the growth medium did not further improve the yield of intact hormone.The effect of5mM EGTA in the growth medium was comparable to5mM EDTA.None of the other protease inhibitors had any significant effect on the yield of intact hor-mone.Hence,metallopeptidases seem to be responsi-ble for extracellular degradation of hPTH,and a four to five times increase in the yield of secreted intact hPTH could be obtained simply by adding EDTA to the pro-duction medium.The growth of the cells was not im-paired by the presence of10mM EDTA in the culture media.
These data clearly show that the present P.pastoris expression systems,especially the Mut S phenotype,are superior to our best S.cerevisiae systems(Vad et al., 1998)with regard to thefinal level of intact hPTH. The appearance of the6-kDa band was however not affected by addition of EDTA,suggesting that a sub-stantial amount of hPTH degradation products is still formed.
3.4.Coexpression of protein disulphide isomerase stimulates the secretion of hPTH
Protein disulphide isomerase(PDI)is an ER-resident foldase exhibiting both isomerase and chap-erone activities.Increasing PDI activity in different yeast expression systems has been reported to improve the secretion of some heterologous proteins contain-ing disulphide bridges(Robinson et al.,1994;Hayano et al.,1995;Shusta et al.,1998;Bao and Fukuhara, 2001).With the intention to elucidate whether the chap-erone activity of PDI accelerates the secretion of small foreign proteins without cystein residues in P.pastoris, we investigated the effect on the hormone yield by co-expression of hPTH and S.cerevisiae PDI.
The hPTH expressing transformant GS115/K23was transformed with a sequence encoding S.cerevisiae PDI under control of the AOX1promoter,and the in-tegration of PDI expression cassettes in the double
R.Vad et al./Journal of Biotechnology116(2005)251–260257 transformants was confirmed by PCR amplification.
The PDI mRNA levels in the double transformants
increased only modestly compared to GS115/K23,
which contains one copy of the PDI1gene(results
not shown).Nevertheless,when the double transfor-
mants were induced in BMMY medium for120h
in the presence of10mM EDTA,the level of intact
hPTH increased to over300mg intact hormone per
liter growth medium(Table1,Fig.2B).The hormone
yield was not enhanced without EDTA in the culture
medium.
3.5.Purification and characterization of the
secreted products
One of the two prominent bands in Fig.2A migrated
as intact hPTH,while the highest mobility band was
suspected to be composed of proteolytic products.To
identify these bands,media containing the secreted
hPTH forms were clarified by centrifugation,and
concentrated using Sepharose F fastflow columns.
The material was further purified by¨AKTA on reverse
phase columns followed by isocratic elutions.The
dominant peak of the resulting chromatograms was
identified as intact hPTH by SDS–PAGE(Fig.3),
N-terminal sequencing and MS,where the measured
value of molecular mass of the secreted product was
near the theoretical value of intact hPTH of9424.3Da.
In addition,the MS analyses revealed that the
band
Fig.3.Silver stained gel(15%)after SDS–PAGE of intact hPTH purified by reverse phase chromatography(lane1)as described in Section2;lane2:PTH standard mixed with molecular size markers.migrating as intact hPTH also contained a minor com-ponent with a molecular mass consistent with the mass of an O-glycosylated form of hPTH(hPTH-glyco1) produced by S.cerevisiae(Olstad et al.,1992).
One of the main components of the other promi-nent band was by N-terminal sequencing identified as ubiquitin.The molecular mass of ubiquitin is8.5kDa, which was confirmed by MS of the isolated compo-nent.Ubiquitin migrated however as a6kDa protein on SDS–PAGE gels.This band appeared only upon in-duction of hPTH expressing cells,and the GS115wild strain did not secrete a corresponding band by growth on methanol.No specific proteolytic products of hPTH were detected.
4.Discussion
The methylotrophic yeast P.pastoris has turned out to be a suitable host organism for production of recom-binant mammalian hormones,and thereby provide ad-equate quantities for structure–function studies.Exam-ples of such hormones are ovine leptin(Laborde et al., 2004),human luteinizing hormone and chorionic go-nadotropin(Gadkari et al.,2003),and tethered ovine follicle-stimulating hormone␤␣(Fidler et al.,2003).
We have previously used S.cerevisiae as host for heterologous production of hPTH.The hormone pro-duction of this yeast was however limited,a maximum of16mg hPTH Q per liter growth medium was obtained after improvements of the expression system(Vad et al., 1998).Hence,the P.pastoris hPTH expression sys-tem producing over300mg authentic hormone per liter growth medium is superior to our best S.cerevisiae system.In both yeast systems,the Kex2protease is re-quired for the proteolytic processing of the MF␣pro-parathyroid hormone precursor.The residues near the recognition site influence the activity of the S.cere-visiae Kex2protease(Rockwell et al.,2002),which possibly has different specificity from the P.pastoris Kex2-like endoprotease since the internal tribasic pu-tative Kex2p cleavage site R25K26K27in hPTH is not exposed to cleavage in contrast to hPTH produced by S.cerevisiae.However,since a Kex2-like protease has been reported to be a problem during protein expres-sion in P.pastoris(Werten et al.,1999;Shapiro et al., 2003),another possibility is that hPTH is protected by its folding or associated with other proteins.
258R.Vad et al./Journal of Biotechnology116(2005)251–260
The level of intact hPTH secreted by P.pastoris was significantly enhanced by the presence of EDTA in the growth medium.Addition of5mM EDTA has been rec-ommended as an approach to stabilize a foreign protein (Sreekrishna et al.,1997),and was shown to increase the production of single-chain antibody several-fold (Shi et al.,2003).Doubling thefinal concentration of EDTA from5to10mM markedly increased the level of intact hPTH,which indicates that principally extracel-lular or cell wall-associated metallopeptidases may be responsible for the instability of the hormone.EDTA, an unspecific chelating agent,inhibits in addition other types of cation-activated peptidases.Such enzymes in-clude among others calpain peptidase,an intracellular Ca2+-dependent cystein protease that is ubiquitously distributed(Barrett et al.,1998).S.cerevisiae has one putative gene for a calpain-like protease(Futai et al., 1999).
Coexpression of Sc PDI in the hPTH expressing cells was the other important factor contributing to the high yield of hPTH.This approach probably reduced the retention of the hormone within the endoplasmic retic-ulum(ER),which often appears to be a bottleneck in the secretory pathway,suggesting that the chaperone activity of the foldase may promote the folding of pep-tides without any cystein residues in P.pastoris.PDI from P.pastoris(Pp PDI)has64%similarity with S. cerevisiae PDI(Warsame et al.,2001).In our group, we have shown that Pp PDI and Sc PDI equally im-prove the secretion of purple acid phosphatase(PAP) from P.pastoris(Blindheim et al.,unpublished).The amino acid residues of PDI important for function seem therefore to be conserved.Although these ob-servations are consistent with the hypothesis that PDI acts as a chaperone independently of the isomerase ac-tivity(Wang,1998),indirect effects on the secretion machinery cannot be excluded.Recently,it was re-ported that overexpression of PDI in S.cerevisiae en-hanced the secretion of␤-glucosidase,containing only one cystein residue,either by acting in a chaperone-like mode or by creating mixed disulfides with the enzymes lone cystein residue during the folding and assembly process(Smith et al.,2003).On the other hand,PDI from S.cerevisiae had in contrast to mam-malian PDI no effect on the renaturation of denatured citrate synthase or glyceraldehyde3-phosphate dehy-drogenase(GAPDH),both enzymes lacking disulphide bonds(Katiyar et al.,2001).
The secretion products from the P.pastoris trans-formants consisted primarily of intact hPTH,but one minor hPTH form with a higher molecular mass was also detected by N-terminal sequencing and MS anal-yses.The mass of the other hPTH form was close to the mass of a biologically active O-glycosylated form of hPTH,substituted with two mannosyl residues at Thr79,reported to be secreted by S.cerevisiae.This yeast produced in addition one more O-glycosylated hPTH form(Olstad et al.,1992).O-mannosylation is a phenomenon that has been observed in several for-eign proteins expressed in P.pastoris(Cereghino and Cregg,2000),but few studies have reported their char-acteristics(Trimble et al.,2004;Boraston et al.,2003).
We observed a high production of ubiquitin in the hPTH producing cells.What causes this is unknown, but the phenomenon could be stress-related.The role of extracellular ubiquitin is not well understood.Inter-estingly,a few studies have reported a positive effect of intracellular ubiquitin expression indicating a link between heterologous protein production and the ubiq-uitin system.In our case,this observation was not fur-ther elucidated.In S.cerevisiae,ubiquitin overexpres-sion has been demonstrated to enhance the secretion of human leucocyte elastase inhibitor seven-fold(Chen et al.,1994),while duplication of the ubiquitin gene in ctis resulted in a striking increase in the secre-tion of recombinant human serumalbumin,but not of human interleukin1␤(Bao and Fukuhara,2001).
In conclusion,we have engineered a P.pastoris hPTH expression system capable of the secretion of over300mg intact hPTH per liter growth medium in shakeflasks in the presence of10mM EDTA com-bined with coexpression of Sc PDI.We believe that it should be possible to develop a strategy for cultivation of the hPTH producing cells in a bioreactor,which de-mands different conditions than in shakeflasks,for the production of sufficient amounts of the hormone for biochemical investigation on this potentially important and complex biopharmaceutical. Acknowledgements
We wish to express our deepest gratitude to Profes-sor Tordis B.Øyen,the supervisor of this project until she died January2002.We also thank Bich Thi Vu for technical assistance during part of this work.。