Laboratoire de Génétique Moléculaire et Cellulaire, INA-PG, INRA UR216, CNRS URA1925, BP01 F-78850 Thiverval Grignon, France1
Aventis Pharma France, 13, quai Jules Guesde BP14, 94403 Vitry sur Seine, France2
Author for correspondence: Dominique Swennen. Tel: +33 1 30815444. Fax: +33 1 30815457. e-mail: swennen{at}grignon.inra.fr
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ABSTRACT |
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Keywords: heterologous secretion, glucoamylase
Abbreviations: AEP, Yarrowia lipolytica alkaline extracellular protease; GST, glutathione S-transferase; Kex2p, Kex2 protease (Kexin, EC 3 . 4 . 21 . 61); scFv, single-chain antibody fragment(s)
a D. Swennen and M.-F. Paul contributed equally to this work.
b Present address: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
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INTRODUCTION |
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Problems encountered with heterologous expression are often linked to the heterologous protein expressed and are particularly observed with scFv proteins. It is thus important to have a range of possible hosts to investigate the best one for efficient scFv production. Yarrowia lipolytica and Kluyveromyces lactis, two non-conventional yeasts, can efficiently secrete heterologous proteins (Gellissen & Hollenberg, 1997 ; van der Berg et al., 1990
; Fleer, 1992
; Muller et al., 1998
; Dominguez et al., 1998
). Both are generally recognized as safe (GRAS) organisms. Y. lipolytica is able to secrete approximately 1 g alkaline extracellular protease (AEP) l-1 into the medium under optimal conditions (Barth & Gaillardin, 1997
), suggesting good potential for secretion of heterologous proteins. The promoter of the XPR2 gene encoding AEP has been used to direct heterologous protein expression in Y. lipolytica (for examples see Park et al., 1997
; Muller et al., 1998
). Regulation of this promoter is very complex thus limiting its industrial use. A hybrid promoter has been constructed, based on tandem copies of upstream activator sequences from the XPR2 promoter (Madzak et al., 1999
). This hybrid promoter (hp4d) is weakly affected by environmental conditions and drives a quasi-constitutive protein expression (Madzak et al., 2000
). Similarly, K. lactis was reported to secrete high-molecular-mass proteins (Wesolowski-Louvel et al., 1996
).
To analyse the scFv secretory capacity of these two yeasts, we have constructed expression vectors that allow secretion of the anti-p21ras scFv (Y28), a single-chain antibody derived from the neutralizing mAb Y13-259 (Werge et al., 1990 ). Expressed genes were placed under the control of the quasi-constitutive promoter hp4d for Y. lipolytica (Madzak et al., 2000
) and of the lactose-inducible LAC4 promoter for K. lactis. Y28 was fused to different pre- or prepro- regions, or to Arxula adeninivorans glucoamylase (glucan 1,4-
-glucosidase, EC 3 . 2 . 1 . 3), which was previously shown to be secreted as an active form in both S. cerevisiae (Bui et al., 1996a
) and K. lactis (Bui et al., 1996b
), thus allowing them to grow on starch-containing medium. This enzyme was used as a reporter of secretion and was separated from the scFv domain by a Kex2 protease (Kex2p; Kexin, EC 3 . 4 . 21 . 61)-like proteolytic site (Contreras et al., 1991
) to allow cleavage of recombinant Y28. Both organisms secreted soluble and functional scFv, with yields depending on the nature of the expression cassette, the highest ones reaching 1020 mg l-1.
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METHODS |
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The Yarrowia lipolytica strain P01f (MATA ura3-302 leu2-270 xpr2-322 axp2-NU49 XPR2::SUC2) (Madzak et al., 2000
) was used as recipient.
The Kluyveromyces lactis strain MW98-8C (Mata uraA argA lysA K+ pKD1) was kindly provided by M. Wesolowski-Louvel (Université Claude Bernard, Villeurbanne, France). Strain FB05 (CBS29 591: patent no. FR9109854; Rhône-Poulenc Rorer SA) was obtained after disruption of the 3-phosphoglycerate kinase gene (Fournier et al., 1990 ) in the CBS1065 background (CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands).
E. coli cells were grown in LB medium (1% bactotryptone, 1% yeast extract, 0·5% NaCl) with 100 µg ampicillin ml-1 at 37 °C. Y. lipolytica cells were cultivated either on rich YPD medium (1% yeast extract, 1% bactopeptone, 1% glucose) at 28 °C or on minimal medium (0·67% yeast nitrogen base without amino acids; Difco) with 0·8% glycerol or 2% starch (Prolabo) as carbon source in 50 mM phosphate buffer, pH 6·8, at 28 °C. K. lactis strains were grown on glucose rich medium (YPD) or on minimal medium (YNB) with 1% starch, 2% glucose or 2% lactose as carbon source and the appropriate amino acids and bases, as described by Rose et al. (1990) . Starch plates were used for halo assays of glucoamylase activity (Tokunaga et al., 1993
). Transformed strains were grown in the presence of G418 (200 mg l-1).
Plasmids
Construction of Y. lipolytica integrative expression vectors.
p0 is a pBluescript derivative containing the Y. lipolytica URA3 gene on an EcoRIHindIII fragment from pINA156 (R. W. Wing & D. M. Ogrydziak, unpublished), the hp4d hybrid promoter of pINA993 (Madzak et al., 2000 ) between ClaI and SfiI as a PCR fragment amplified with oligonucleotides 5'-CCATCGATAGGCTCTCAAGGGCATCGGTC-3' and 5'-GGATCTGGCCACTGCGGCCTGTGGATGTGTGTGGTTGTATG-3', a polylinker (SfiI, BamHI, EcoRI, NdeI, NotI) obtained by annealing 5'-TGGCCGGATCCGAATTCCATATGGC-3' and 5'-GGCCGCCATATGGAATTCGGATCCGGCCACTG-3', and a NotIKpnI fragment containing an XPR2 terminator engineered to contain an AscI site obtained by PCR from pBUPAEP (D. Swennen, unpublished) with 5'-AAAAAGGAAAAGCGGCCGCGTCTACTCCGGGACCTCCCTGGC-3', 5'-AATGCCAACACCGTTGTAGGCA-3', 5'-GGGCGCGCCTAGGCAATTAACAGATAGTTTGCC-3' and 5'-GGGGTACCTATGGAAAATAAGAAATACGACAGT-3'. Thus, targeted integration into the XPR2 terminator can be obtained after AscI digestion of the construct.
PGAA is a p0 derivative containing a BamHIBamHI fragment from pBscG/GAA/C (provided by K. D. Breunig, Martin Luther Universität, Halle, Germany) encoding glucoamylase and a SfiI/BamHI linker sequence (oligonucleotides 5'-TGGCCG-3' and 5'-GATCCGGCCACTG-3').
pPY28 is another p0 derivative containing a Y28 fragment (patent no. WO94/29446; Rhône-Poulenc Rorer SA) amplified with oligonucleotides 5'-GGGAATTCTGCGCACAGGTGCAGCTGCAGGAG-3' and 5'-GGAATTCTTATTAATTCAGATCCTCTTCTG-3', digested with FspI and EcoRI, and a synthetic fragment encoding the XPR2 signal sequence using oligonucleotides 5'-TGGCCATGAAGCTCGCTACCGCCTTTACTATTCTCACTGCCGTTCTGGCC-3' and 5'-GGCCAGAACGGCAGTGAGAATAGTAAAGGCGGTAGCGAGCTTCATGGCCACTG-3'.
pPPY28 results from the insertion into p0 of the same FspIEcoRI Y28 fragment and of a SfiIStuI PCR fragment encoding the XPR2 prepro-sequence amplified from pBUPAEP using oligonucleotides 5'-AAGAGCACCAACCCACT-3' and 5'-AAGGCCTCTTGGCATTAGAAGAAGCAGG-3'.
pGAA-Y28 results from insertion into p0 of a SfiIHindIII fragment from pGAA and a HindIIINotI fragment from the K. lactis pKNH5 plasmid.
Construction of K. lactis replicative expression vectors.
The deoxyribonucleotides 5'-AGCTTGGGTTAATTAAGGGGGCCGGCCCTAGGCGGCCGC-3' and 5'-AGCTGCGGCCGCCTAGGGCCGGCCCCCTTAATTAACCCA-3' were paired and ligated into the HindIII site of the replicative pKD-1-based expression vector pYG1043 (Fleer et al., 1991 ), derived from pYG1023 (patent no. FR9109854; Rhône-Poulenc Rorer SA), to introduce three unique restriction sites (PacI, FseI and NotI) and a stop codon between the FseI and NotI sites, generating pKNH18. The gene encoding glucoamylase was amplified from pBscG/GAA/c by PCR with the primers 5'-GGGTTAATTAAAAACAATGCGTCAGTTTCTAGCACTTGCTG-3' and 5'-CCCGGCCGGCCTAGGTCAGCAACATTGGTGAGATAG-3', and cloned into pKNH18 as a PacIFseI fragment, generating pKNH6.
The sequence encoding the fusion of the Kex2p-like site and Y28 was amplified by a two-round PCR using the scFv sequence as template with two forward primers, 5'-GTCATCTCCAAGCGGATGGCCCAGGTGCAGCTGCAG-3' and 5'-GGGGGCCGGCCGAACGTCATCTCCAAGCGGATGGCCCAG-3', and the reverse primer 5'-CCCGCGGCCGCCTACCGTTTGATTTCCAGCTTGGTGCCAGC-3' to clone the chimeric sequence as a FseINotI fragment into pKNH18, generating pKNH23. The FseINotI fragment from pKNH23 was cloned into the glucoamylase-encoding plasmid pKNH6, generating pKNH5, encoding the fusion between the glucoamylase gene and the Y28 gene (patent no. WO94/29446; Rhône-Poulenc Rorer SA), separated by the Kex2p-like encoding sequence.
The sequence encoding Y28 devoid of the Kex2p-like site was made by PCR amplification using the forward primer 5'-GGGGGCCGGCCGATGGCCCAGGTGCAGCTGCAG-3' and the reverse primer 5'-CCCGCGGCCGCCTACCGTTTGATTTCCAGCTTGGTGCCAGC-3' to clone the Y28 sequence as a FseINotI fragment into pKNH18, generating pKNH33, and into pKNH6, generating pKNH1.
The glucoamylase signal sequence (Bui et al., 1996b ) was amplified by PCR using pKNH6 as a template with the primers 5'-GGGTTAATTAAAAACAATGCGTCAGTTTCTAGCACTTGCTG-3' and 5'-CCCCCCCGGCCGGCCTGCCACCGCTATGGAAGCAGCAGC-3', and was cloned as a PacIFseI fragment into pKNH23 and pKNH33, generating pKNH1ss and pKNH11ss, respectively.
Glutathione S-transferase (GST) fusions.
cDNA encoding the C-terminal part of ß-amyloid precursor (cAPP, aa 650695) (negative control) and the full length Harvey-RasVal12 (HaRasVal12) were cloned into pGEX4T1 (Pharmacia).
DNA techniques.
Standard techniques have been used according to Sambrook et al. (1989) . Enzymes were supplied by Gibco-BRL Life Technologies and New England Biolabs. All vectors were checked by sequencing on a fluorescent DNA sequencer (ABI Biosystems Perkin-Elmer) according to the supplier.
Transformation procedures.
E. coli strains were transformed by the method of Chung & Miller (1988) . Y. lipolytica strain transformations were carried out according to Xuan et al. (1990)
. K. lactis strains were transformed by the method of Dohmen et al. (1991)
.
Protein determination.
Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad) or by the method of Lowry using bovine serum albumin as standard.
Solubilization of Y28 inclusion bodies.
E. coli BL21(DE3) cells transformed with the pet29a (Novagen)-Y28 plasmid were grown at 28 °C. Fusion protein synthesis was induced by adding 0·5 mM IPTG to the culture medium and incubating for 3 h. Cells were collected and lysed by sonication in 10 mM Tris/HCl, pH 8, 1 mM EDTA, 1 mM PMSF buffer and the pellet was washed three times in the same solution. Inclusion bodies were solubilized in 4 M urea, 10 mM DTT, 10 mM Tris/HCl, pH 8, 1 mM EDTA, 1 mM PMSF, for 2 h at 28 °C with gentle agitation or in 0·6% SDS. Solubilized proteins from the supernatant after 15 min centrifugation at 10000 g were analysed by Western blotting and the protein concentration was measured.
Protein analysis by Western blotting.
Aliquots from yeast cultures were centrifuged (450 g). PMSF (1 mM) was added to supernatant and cells were washed and resuspended in 0·5 ml 10 mM Tris/HCl, pH 8, 1 mM EDTA, 1 mM PMSF, and lysed by TCA precipitation. Samples were subjected to SDS-PAGE analysis (homogeneous 10% acrylamide or 420% Tris-glycine Novex gels) using the buffer system of Laemmli (1970) . Proteins were detected by Coomassie blue staining or transferred onto a nitrocellulose membrane (Protran BA 85; Schleicher & Schuell) and subjected to immunodetection by a standard procedure (Sambrook et al., 1989
). Rabbit serum directed against scFv was kindly provided by Dr J. L. Teillaud (INSERM U 255, France). Rat monoclonal anti-Ras antibody Ab-1 (clone Y13-259) was from Calbiochem. Horseradish-peroxidase-labelled secondary antibodies were obtained from Interchim and phosphatase-conjugated antibodies from Promega.
Proteinprotein interaction assays.
GST fusion proteins were produced in E. coli BL21(DE3), resuspended in TE buffer (10 mM Tris/HCl, pH 7·5, 1 mM EDTA) containing 0·2% Triton X-100, then bound to glutathione agarose (Sigma) following standard procedures (Smith & Johnson, 1988 ). Supernatant from a 120-h-old rich medium culture of Y. lipolytica carrying either pPY28, pPPY28 or p0 [mixed with v-H-ras (Ab-1) mAbs (Calbiochem) as positive control] was mixed with GSTRas or GSTcAPP glutathione agarose in equal volumes. Proteins present in medium from transformed K. lactis cultures were ethanol-precipitated or filtered onto a Sephadex G25 column (Pharmacia) equilibrated in TE buffer before mixing with GST fusions. Samples were incubated with 100 µl GST fusions bound to agarose beads for 1 h at room temperature in PBS buffer (50 mM Na2HPO4, 10 mM KCl, 1 mM MgSO4, pH7) containing 0·2% Triton X-100. Beads were washed with 30 column volumes of PBS buffer and 0·2% Triton X-100. Bound proteins were eluted from the resin by heating the samples in SDS-PAGE loading buffer at 95 °C for 10 min. Proteins from equivalent volumes of total, unbound and bound fractions were separated by SDS-PAGE, then subjected to immunodetection.
Glucoamylase assay.
Aliquots of supernatant cultures were incubated in 40 mM citrate buffer (pH 4) containing 0·8% soluble starch (Prolabo) in a final volume of 0·5 ml. After incubation at 50 °C for 20 min, the reaction was stopped as described by the manufacturer by addition of 1 ml glucose (trinder) reagent (Sigma) to quantify the released amount of glucose (modified from Büttner et al., 1987 ). The halo assay was done on yeast cells grown on starch-containing medium for 27 d. Haloes surrounding the transformants were detected by the absence of iodo-staining (I2/KI) of the hydrolysed starch (Tokunaga et al., 1993
).
N-terminal sequence determination.
Automated N-terminal Edman sequencing was performed using a Perkin Elmer Applied Biosystems Procise 494A sequencer with reagents and methods of the manufacturer. Supernatants were ethanol-precipitated (70% final ethanol). Samples were separated by SDS-PAGE electrophoresis and transferred onto SequiBlot PVDF membrane (Bio-Rad). Proteins were analysed at the picomole level.
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RESULTS |
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Characterization of secreted Y28
Y. lipolytica. To get information about Y28 protein structure, aliquots of culture supernatants of strains carrying pPY28 and pPPY28 were analysed by SDS-PAGE in the presence or absence of ß-mercaptoethanol. Western blotting with anti-Y28 serum revealed that oxidized Y28 migrated slightly faster than reduced Y28, indicating the probable formation of disulfide bonds (not shown). N-terminal sequencing of Y28 proteins produced by Y. lipolytica strains transformed with pPY28 and pPPY28 indicated in both cases that cleavage occurred at the expected position (not shown), either after the usual signal peptide cleavage site or at the Kex2p-like site. In vitro assays showed that Y28 produced by Y. lipolytica was able to bind the antigen bound to glutathione agarose as did the mAb from which Y28 originates (Fig. 4). As expected, neither the Y28 proteins nor the mAb were retained on the GSTcAPP fusion agarose beads.
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Y28 proteins secreted by Y. lipolytica (transformed with integrative plasmids pPY28 or pPPY28) and K. lactis (FB05 transformed with the replicative plasmid pKNH5) were compared by SDS-PAGE analysis and Western blotting with anti-Y28 serum (Fig. 7). The Y28 protein secreted by K. lactis had a lower molecular mass than the scFv produced by Y. lipolytica because of the absence of the myc-tag. Comparison of the signals to those of known amounts of scFv produced in E. coli as inclusion bodies (not shown) allowed us to estimate that Y28 concentration was 20 mg l-1 for Y. lipolytica and 10 mg l-1 for K. lactis, in shake flask culture, corresponding to 2 and 1% of total cell proteins, respectively.
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DISCUSSION |
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K. lactis strains MW98-8C and FB05 transformed with constructs encoding Y28 failed to grow on expression-inducing medium, suggesting that Y28 overexpression was toxic to cells. Similarly, transformants selected for enhanced copy number of the expression plasmid in Y. lipolytica, as described by Le Dall et al. (1994) , led to severe loss of viability (unpublished). Even though the reasons for scFv toxicity are unknown, attempts at improving the production yields are under way. The use of a promoter inducible in late stationary phase could be more appropriate for anti-Ras scFv protein production. Overexpression of chaperone/foldase has been shown to improve secretion levels in S. cerevisiae (Shusta et al., 1998
) and overexpression in Y. lipolytica of Kar2p (Lee & Ogrydziak, 1997
) and Sls1p (Boisrame et al., 1996
), two endoplasmic resident proteins that are known to interact for efficient co-translational translocation of secreted proteins (Boisrame et al., 1998
) might have similar effects.
More generally, improving fermentation conditions may significantly increase secretion yields: using similar constructs and alternative growth conditions enhances chymosin secretion in Y. lipolytica from 20 to 160 mg l-1 (Madzak et al., 2000 ). Although some differences in secretion efficiency were observed between both yeasts, their secretion capacity for the proteins analysed is approximately equivalent and indicates that these two non-conventional yeasts may be considered as valuable alternative hosts to secrete recombinant scFv.
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ACKNOWLEDGEMENTS |
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Received 18 June 2001;
revised 12 September 2001;
accepted 1 October 2001.