Expression of CFP32 in Pichia pastoris RESEARCH
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High Level Expression of Recombinant Mycobacterium tuberculosis Culture Filtrate Protein CFP32 in Pichia pastoris C. Benabdesselem,1, 2 M. R. Barbouche,2 M. A. Jarboui,1 K. Dellagi, 2 J. L. Ho,3 and D. M. Fathallah1,* Abstract Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc(His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential. Index Entries: Mycobacterium tuberculosis; recombinant; culture filtrate protein; Pichia pastoris.
1. Introduction The release of the Mycobacterium tuberculosis complete genome sequence (1) as well as data from its proteome analysis have urged the evaluation of the pathogen’s culture filtrate proteins as candidate targets for vaccine or diagnostic development (2). Originally called Rv0577, CFP32 is one of the 30 culture filtrate (CF) proteins that have emerged as potential candidates for the serological diagnosis of tuberculosis (TB) (3). The gene encoding CFP32 is found exclusively in the M. tuberculosis complex, as assessed by polymerase chain reaction (PCR) and Southern
blot analysis of the genomes of several mycobacterial strains (4). CFP32 was identified by mass spectroscopy, N-terminal sequencing, and immunodetection (5,6). CFP32 is expressed by M. tuberculosis complex (MTC) members including Bacille Calmette Guerin (BCG) (7). Native CFP32 was detected in the sputum of patients with active pulmonary TB (7). Furthermore, the virulence-related neutral red character, typical of virulent mycobacteria, was recently shown by Andreu et al. (8) to be associated with the cfp32 gene with gene transfer into Mycobacterium smegmatis. Moreover CFP32 levels in the lungs of active pa-
*Author to whom all correspondence and reprint requests should be addressed. 1Molecular Biotechnology Group, 2Cellular and Molecular Immunology Group, Laboratory of Immunology, Institute Pasteur de Tunis, BP74, 1002, Tunis, Tunisia; and 3Division of International Medicine and Infectious Diseases, Department of Medicine, Joan and Sanford I. Weill Medical College of Cornell University, New York, NY, E-mail:
[email protected],
[email protected]. Molecular Biotechnology 2006 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN: 1073–6085/Online ISSN: 1559–0305/2007/35:1/041–050/$30.00
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tients with TB were positively correlated with amounts of the immunosuppressive cytokine interluekin (IL)-10 (7). These observations suggest that CFP32 may play a role in TB pathogenesis and likely interact with the immune system, as previously shown by the detection of serologic reactions to Escherichia coli-expressed rCFP32 in approx 30% of patients with TB (7). Considerable efforts have been made in the identification of antigens that allow the development of a rapid test for the serodiagnosis of TB. These efforts have been hindered by difficulties in producing and purifying recombinant M. tuberculosis antigens in E. coli mainly because of poor yield, insolubility, and gene instability (9–12). E. coli genes have low GC content and probably lack the transcriptional and translational machinery needed to efficiently express mycobacterial genes that have high GC content (13). Expression of M. tuberculosis antigens in E. coli was enhanced after replacement of low-usage codons; however, the highest yield achieved was still only 80 mg/L of culture for mycobacterium Ag85A (14). Furthermore, procedures for engineering Mycobacterium genes are cumbersome and lengthy. The difficulties in overexpressing mycobacterial antigens in E. coli has led investigators to use other expression systems, such as baculovirus (15,16), Streptomyces lividans, and Corynebacterium spp. (17–19). Closely related nonpathogenic mycobacterium, such as M. smegmatis and BCG (20–22), were also used to express mycobacterial genes by heterologous complementation. However, these are not conventional systems for the production of high levels of recombinant proteins. It is also known that heterologous complementation of MTB genes into BCG or M. smegmatis may be associated with gene modifications and loss of protein activity. Furthermore, these hosts will need to be manipulated in level 3 standard biosafety containment if transfected with virulence-associated genes. Hence to provide the large amounts of mycobacterial proteins needed for diagnostic evaluation or large-scale immunization, more efficient expression systems need to be explored.
The methylotrophic yeasts, such as Pichia pastoris, are known for their ability to produce high amounts of recombinant proteins (23–27). P. pastoris has the ability to efficiently produce posttranslationally modified proteins and has a high GC-rich preferred codon usage (28,29), suggesting that improved transcription and translation of mycobacterial genes could be achieved. However, the use of this expression system for the expression of mycobacterial proteins, and especially those having serodiagnostic or vaccine potential, has not been explored. In this report, we describe the high yield production of mycobacterial proteinsin P. pastoris, and the characterization of a recombinant form of M. tuberculosis culture filtrate protein CFP32.
2. Materials and Methods 2.1. Plasmids The plasmid pPICZαA, purchased from Invitrogen (Invitrogen Corporation, Carlsbad, CA), was the vector used to transfer the CFP32 expression cassette into P. pastoris. CFP32 cDNA was propagated using plasmid pQE31/Rv0577 as the template.
2.2. Strains E. coli Top 10F' was used in all subcloning steps. P. pastoris KM71H strain (Invitrogen) was used to express rCFP32.
2.3. Construction of CFP32 Expression Cassette The cDNA encoding the mature CFP32 was amplified by PCR using plasmid pQE31/Rv0577 as the DNA template (4) and the following specific primers: forward 5'-CCGGAATTCCCC AAGAGAAGCGAATACAG-3' and reverse 5'GCTCTAGAGCTTGCTGCGGTGCGGGCTT-3'. The primers were designed to include an EcoRI site at the 5' end of the forward primer and an XbaI site at the 5' end for the reverse primer. The latter also included an additional alanine codon at the 3' end. The PCR products were purified on a PCR prep column (Qiagen, Valencia, CA) and digested using EcoRI/XbaI. After purification, the amplified DNA was ligated to the transfer vector
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Expression of CFP32 in Pichia pastoris pPICZαA, downstream of the secretion signal of the alpha matting factor from Saccharomyces cerevisiae under the control of the alcohol oxidase 1 promoter (AOX1). The resulting expression cassette was verified by DNA sequencing. Standard techniques (30) were used for DNA modification, ligation, and plasmid transformation. Restriction endonucleases and other enzymes were used as recommended by the supplier (Invitrogen).
2.4. Transformation of P. pastoris The recombinant plasmid pPICZα-Rv0577 was propagated using the E. coli Top 10F'. Plasmid DNA was isolated from selected transformants, digested with BstXI restriction enzyme (Amersham Biosciences, Uppsala, Sweden) and used to transform P. pastoris KM71H by electroporation.
2.5. Analysis of Genomic DNA To check for the integration of the expression cassette into the P. pastoris genome, genomic DNA of a number of transformants was analyzed by PCR. The isolation of genomic DNA and PCR amplification was then carried out according to the Pichia manual (Invitrogen). Primers complementary to the 5' and 3' region of the Aox 1 gene were used for PCR amplification. DNA sequencing was performed using a conventional Big Dye Terminator cycle sequencing ready reaction kit (Perkin Elmer, Applied Biosystems, Foster City, CA) and an ABI373 automated DNA sequencer.
2.6. In Silico Amino Acid Sequence Analysis The following software and databases were used to analyze CFP32 amino acid sequence: ScanProsite version 99.07 on Prosite at http:// www.expasy.ch/prosite/, Blast Version 2000.1 on ProDom at http://www.protein.toulouse.inra.fr/ prodom.html, PROFsec and PROFacc version 2000_04 at http://cubic.bioc.columbia.edu, and GLOBE version 1. 98.05 at http://www.cubic. bioc.columbia.edu/predictprotein.
2.7. Expression of rCFP32 The recombinant P. pastoris clone was inoculated into 5 mL of yeast extract, bactopeptone, dextrose (YPD) media with 5 µg of Zeocin and incubated at 30ºC in a orbital shaker at 250 rpm
43 overnight. The culture was transferred into 250 mL of Buffered Glycerol Complex Media (BMGY;1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6, 1.34% yeast nitrogen base [YNB], 4 × 10–5% biotine, 1% glycerol) in a 2-L baffled flask and incubated at 30ºC in a shaking incubator at 250 rpm overnight. Cells were harvested through centrifugation at 1200g for 10 min, then resuspended in 25 mL of Buffered Methanol Complex Medium (BMMY; 1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6, 1.34% YNB, 4 × 10–5 biotine, 1% methanol), and incubated for another 72 h with the addition of 1% methanol after every 24 h to maintain induction conditions. Cultures were centrifuged at 1200g for 10 min and the supernatant was collected.
2.8. Protein Electrophoresis Ten microliters of culture supernatant or purified proteins were heated for 5 min at 100ºC after the addition of 5 µL of 3X loading buffer, (125 mM Tris-HCl, pH 6.8, 6% SDS, 0.2% bromophenol blue, and 15% glycerol) and subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-protean II system (Bio-Rad Laboratories, Hercules, CA). An RNP 800 molecular size marker (Amersham Biosciences) was used to measure protein mobility. All gels were run at 120 V, and the proteins were visualized by Coomassie brilliant blue G-250 staining.
2.9. Western Blotting Proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences) using a Multiphor II Electrophoresis System (Bio-Rad Laboratories). The transblotted membrane was blocked with phosphate-buffered saline (PBS)/ 5% fat milk/0.1% Tween-20 (PBS-T-Fat milk) overnight at 4°C. Two types of Western analysis were performed. In the first, a monoclonal antimyc HRP antibody (Invitrogen, R951-25) was used; for the second, the membrane was probed with a rabbit polyclonal antibody directed against CFP32. To detect the fusion protein, the membrane was incubated for 1 h with the anti-myc-HRP
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antibody and then incubated for 1 min with ECL solution (Amersham Biosciences). For visualization of the immunoreactive band with the rabbit antisera, an antirabbit HRP conjugate (Dako, Glostrup, Denmark) was used.
sequencing was carried out by performing 12 cycles of automated Edman degradation in an Applied Biosystems 476 A protein sequencer. Samples were deposited onto Biobrene-precycled glass-fiber discs.
2.10. Purification of rCFP32
2.13. ELISA
A chelating Sepharose Fast Flow column with nickel that selectively retains proteins containing a histidine tag (Amersham Biosciences) was used. Histidine-tagged rCFP32 was eluted using buffers containing imidazole. Briefly, 2 mL of chelating Sepharose Fast Flow (Amersham) were loaded onto a column and washed with 10 mL of distilled water and mixed end-over-end for 5 min. One milliliter of 0.1 M NiSO4 was added and incubated for 10 min at which time the column was equilibrated with 10 mL of start buffer (20 mM Na2HPO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4). Ten-milliliter volumes of culture supernatant were incubated for 1 h with mixing end-over-end rotation at room temperature. One-milliliter volume of elution buffer (20 mM Na2HPO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4) was added and mixed by end-over-end rotation for 15 min. The elution step was repeated a total of five times.
The recombinant CFP32 was immobilized in a 96-well enzyme-linked immunosorbent assay (ELISA) plate overnight at 4°C (2 µg per well) and incubated with serum from patients with TB, diluted 1:200 in PBS. The bound immunoglobulins were detected by adding 100 µL of horseradish peroxidase (HRP)-conjugate sheep antihuman IgG. Binding was revealed by the addition of 150 µL of 1 mg/mL OPD substrate. The plates were incubated for color development then blocked with 3 N sulfuric acid and the absorbances were determined at 492 nm with a microtiter plate reader.
2.11. Protein Quantification The amount of the purified fusion protein CFP32-myc-(His)6 was quantified after dialysis for 48 h against PBS, using a protein kit from Sigma (Sigma, St. Louis, MO) according to the manufacturer’s instruction.
2.12. NH2-Terminal Sequencing High-performance liquid chromatography (HPLC) purification of rCFP32 was performed using a Beckman Series 125 pump and a Beckman diode array detector set at 214 and 280 nm, controlled by the GOLD software. Fractions were loaded onto a C8 reverse-phase HPLC column (5 µm, 4.6 250 mm; Beckman Coulter, Fullerton, CA). The protein was eluted from the column at 1 mL/min in a gradient of 0.1% trifluoroacetonitrile (TFA)/acetonitrile from 10 to 80% in 60 min and fractions 17 and 18 were collected at 39.1 min and 39.6 min, respectively. NH2-terminal amino acid
3. Results 3.1. Clone Design and Expression of Recombinant CFP32 in P. pastoris To use the methyltrophic yeast P. pastoris for the production of a secreted recombinant form of the M. tuberculosis culture filtrate protein CFP32, the specific cfp32(Rv0577) cDNA and plasmid pICZα were used to design an expression cassette. The later consisted on the cloning of the cDNA downstream of the S. cerevisiae α mating factor prepropeptide as a secretion signal and under the control of the P. pastoris AOX1 strong promotor and upstream of the sequence coding for the myc epitope-(His)6 tag. To conserve an open reading frame with the tag, we inserted an additional codon specific for alanine (A) at the 3' end of the cfp32 cDNA. For cloning purposes, two additional residues, glutamic acid (E) and phenylalanine (F) were also inserted at the junction of the α mating factor and the CFP32 amino acid sequence. This expression cassette was inserted into the P. pastoris strain KM71H genome by homologous recombination. We selected three clones that were resistant to 2000 µg/mL of zeocin and retained the highest expressing one for
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the production of rCFP32 in shaker flasks. The SDS-PAGE analysis of the yeast rCFP32 showed an apparent molecular weight of 35 kDa, which is 7 to 8 kDa larger than the theoretically calculated one (27.3 kDa) and 3 to 4 kDa larger than the native CFP32 as estimated by two-dimensional mapping of short-term culture filtrate of M. tuberculosis H37Rv (5).
3.2. Production of rCFP32 in Shake Flask and Purification The recombinant P. pastoris clone with the highest level of CFP32 expression was used for production in shaker flasks containing complex media. The optimal induction time was determined to be 48 h. The concentration of the rCFP32 in the culture supernatant was high enough to allow direct purification in a single-step, using a nickel column. The protein was eluted as a single peak at 500 mM imidazole concentration, and was seen as a single band with a diffuse staining pattern when analyzed by SDS-PAGE (Fig. 1). Interestingly, we obtained approx 0.5 g of purified rCFP32 from 1 L of shaker flask culture supernatant without carrying out any optimization either in the culture or in the purification steps.
Fig. 1. Control of the purified rCFP32 myc-(His)6 fusion protein. Recombinant protein secreted in the culture media was purified using a Chelating Sepharose Fast Flow Nickel column. The rCFP32 was eluted from the column using an imidazole concentration of 500 mM and 10 µL of each fraction run on an SDS-PAGE. Lane 1: culture supernatant before purification; the dashed arrow shows the rCFP32 that has lost the myc-(His)6 tag. Lane 2: flow trough, the band corresponds to the rCFP32 missing the tag. Lane 3: fraction obtained after washing with 20 mM of imidazole. Lanes 4 to 7: elution fractions. M: molecular size marker RNP800 (Amersham).
3.3. Immunological Characterization To confirm the identity of the recombinant protein, Western blot analysis was performed and showed (Fig. 2) that the purified rCFP32 reacted strongly with an anti-myc monoclonal antibody and with a rabbit polyclonal antibody. This antiserum was raised against rCFP32 produced in E. coli as a (His)6 fusion protein. As shown in Fig. 1, lane 1 and Fig. 2, a lower band appears consistently and its intensity increases over time. This band reacts with the anti-rCFP32 sera, but not with the anti-myc monoclonal antibody. This band likely corresponds to the recombinant CFP32 that has lost the myc-(His)6 tag as a result of spontaneous cleavage or proteolysis, a feature that we have already observed with fusion proteins produced in P. pastoris KM71H (31). Furthermore, a preliminary ELISA study using rCFP32 produced in P. pastoris and seven sera
Fig. 2. Expression of the CFP32 gene in yeast (Pichia pastoris). Heterologous expression of the CFP32 gene by pPICZ.577-transformed yeast as shown by Western blot. Ten nanograms of purified yeast rCFP32 were run on 15% SDS-PAGE transferred to nitro cellulose and revealed using: (A) rabbit anti-rCFP32 produced in E. coli as primary antiserum (dilution 1:2000). The minor band shown by the arrow corresponds to the rCFP32 missing myc-(His)6. (B) anti-myc HRP mAb (Invitrogen, R951-25) (dilution 1:5000).
from TB patients showed a strong reactivity compared to four healthy BCG-vaccinated individuals (Fig. 3).
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Benabdesselem et al. ferent types of protein analysis software for the presence of post-translational modification motifs. As shown in Table 1, several putative sequences of post-translational modification were identified. Glycosylation and acylation usually occur in P. pastoris. Prediction of solvent accessibility (core/ surface ratio) indicates that 57.47% of rCFP32 have more than 16% of their surface exposed. These data are confirmed by the prediction of the protein secondary structure, which shows 17% of the protein folds as alpha helixes, 31% as extended sheets, and 52% as loops. Furthermore, prediction of protein globularity showed that rCFP32 appears as compact as a globular domain.
Fig. 3. Box-and-whiskers (mean value ± 2 SD) illustration of the serological recognition pattern of rCFP32 (boxes) by the sera of seven patients with TB and four BCG vaccinated healthy controls (p = 0.06).
3.4. Amino-Terminal Sequencing N-terminal sequencing of HPLC purified rCFP32 was performed to determine whether cleavage of the S. cerevisiae α mating factor signal peptide was properly processed. A single N terminus end was found and corresponded to the Glu-Ala-Glu-Phe-Pro-Lys-Arg-Ser-Tyr-Arg-Gln sequence. The four amino acids (Glu-Ala-GluPhe) at the N terminus correspond to the two last residues of the α factor (Glu-Ala) followed by the two residues (Glu-Phe) introduced at the cloning site. Proline is the first residue of CFP32 originally expressed by Mycobacterium. These data show that the signal peptide is properly cleaved and that the Ste13 protease of the P. pastoris secretory pathway is responsible for the removal of this signal peptide.
3.5. In Silico Analysis of the rCFP32 Amino Acid Sequence To relate the higher size observed for the rCFP32 to potential post-translational modification, the sequence corresponding to the recombinant form of CFP32, including the extra NH2 Glu-Ala-Glu-Phe residues and the extra Ala at the COOH terminus, was analyzed using several dif-
4. Discussion To investigate the capacity of yeast to produce large yields of recombinant M. tuberculosis complex-specific culture filtrate proteins for the assessment of their potential for the serodiagnostic of TB and their relevance to the pathogenesis of TB, we have generated a P. pastoris clone that produces a high level of rCFP32 as a myc-(His)6 tagged protein. Because of cloning constraints, we designed an expression cassette that introduced two extra residues, glutamic acid and phenylalanine, at the NH2 terminus of the molecule as well as an alanine at the COOH end. Alanine was chosen because it is a neutral amino acid that is unlikely to affect the proper folding of the protein. Purification was carried out in a single step and the average yield obtained was 0.5 g/L of culture supernatant. This is a high production yield considering that the culture was performed in shaker flasks and that no specific optimization procedure was carried out in any of the production steps. The higher molecular weight observed for the rCFP32 produced in P. pastoris is probably the result of the production of this molecule as a fusion protein containing a myc-(His)6 tag at the COOH end. However, the predicted size of the myc-(His)6 tag cannot account for the size difference of the recombinant CFP32 produced in P. pastoris. Yeast-specific post-translational modifications probably contribute to the size difference as suggested by the diffused banding pat-
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Table 1 Putative Posttranslational Modification Sites Present in the Amino Acid Sequence of Mycobacterium tuberculosis Culture Filtrate Protein CFP32 Motif ID
N-glycosylation
PKC-phosphorylation
CK2-phosphorylation
N-myristoylation
Consensus
N [^P] [ST] [^P]
[ST] · [RK]
[ST] · {2} [DE]
G [^EDRKHPFYW] · {2} [STAGCN] [^P]
151 TKD
170 SSME
46 GGVYSM 68 GAPEGM 121 GAAVGL 166 GLTHSS 226 GQVIAE 249 GAIFSV
Sequence & Position in rCFP32 139 NETG
PKC, protein kinase C; CK2, casein kinase II. Single letter amino acid nomenclature is used. One glycosylation and two phosphorylation sites were identified. Six acylation (myristoylation) motifs are scattered throughout the sequence.
tern observed in SDS-PAGE. The identification of putative acylation and glycosylation sites in the CFP32 amino acid sequence argue in favor of post-translational modification of the recombinant form produced in P. pastoris. N-terminal sequencing suggested that cleavage of the α mating factor signal peptide was essentially carried out by the Ste13 protease. The S. cerevisiae α mating factor prepropeptide can be cleaved at different sites by two proteases, namely Kex2 and Ste13, and variations of cleavage have been observed for a number of recombinant protein expressed in P. pastoris (29,31). Proper cleavage of the heterologous signal peptide is important to yield a recombinant protein that is as close as possible to the native protein. Recombinant CFP32 produced in P. pastoris is recognized by sera from patients with TB, suggesting that yeast rCFP32 is similar to the native CFP32 and thereby has immunogenic epitopes. These immunogenic epitopes could be the result of either protein folding nearly identical to that of the native CFP32 or posttranslational modifications, which are common in mycobacterial antigens. Identification of a number of putative motifs for post-translational modifications in the CFP32 amino acid sequence, which can be performed by P. pastoris (23,24), also argue in favor of this possibility. The high solvent accessibility index
observed for rCFP32 is an indicator of increased antigenicity and could explain the good reactivity of rCFP32 with the sera from the patients with TB (100% in our preliminary study as compared to the 30% previously reported with rCFP32 produced in E. coli) (7). The rCFP32 produced in P. pastoris could be very useful in conducting further analysis of the protein’s structure–function, and mainly its potential role in the pathogenesis of TB as suggested earlier (7,8). Furthermore, reactivity of rCFP32 with the sera from the patients with TB is being investigated with a larger panel of patients (Ben Abdessalem et al., in preparation). Our findings underscore the potential of using the yeast P. pastoris to produce high amounts of recombinant mycobacterial antigens that structurally approximate the native protein. By overcoming the obstacle of producing large quantities of mycobacterium antigens, mainly those found in culture filtrate, further investigation of their vaccine or diagnostic potential, as well as their role in the pathogenesis of the disease will be facilitated.
Acknowledgments We thank Dr. N. Srairi for her assistance in determining the rCFP32 NH2-terminal sequence. We are grateful to H. Merdassi for providing patient sera.
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Expression of CFP32 in Pichia pastoris 25. Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast. Yeast 8, 423–488. 26. Sreekrishna, K., Brankaamp, R. G., Kropp, K. E., et al. (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190, 55–62. 27. Wendeler, M., Hoerschemeyer, J., John, M., et al. (2004) Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labelling, and biophysical characterization. Prot. Expr. Purif. 34, 147–157. 28. Scorer, C. A., Buckholz, R. G., Clare, J. J., and Romanos, M. A. (1993) The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene 136, 111–119.
49 29. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249–270. 30. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 31. Guedel-Ben Tanfous, N., Kallel, H., Jarboui, M. A., and Fathallah, D. M. (2005) Expression in Pichia pastoris of a recombinant scFv form of MAb 107, an anti human CD11b integrin antibody. Enzymol. Microb. Technol. 38, 636–642.
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