Construction,production, and characterization of recombinant scFv antibodies against methamidophos expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos for downstream applications.     

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l⁻¹ in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis>

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence α-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3′-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

High-Level Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, Purification and Mosquito-Larvicidal Activity of the Binary Toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis>

The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin, the binA and binB genes were separately cloned in Eschericia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity of the fusion protein.     

Changing the N-terminal sequence protects recombinant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> circumsporozoite protein from degradation in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) _n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The administration of antibodies against the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a promising approach in the upregulation of immune responses in many cancers and infectious diseases. The single-chain variable fragment of antibody against CTLA4 is also useful in developing immunotoxins that might be used in the treatment of cancer, transplant rejection, and autoimmune diseases. Here, we report the production of a soluble and functional scFv antibody against CTLA4 by using Pichia pastoris as the expression system. The gene encoding scFv hS83 with an additional 6His-tag at the 5’-end was inserted into the expression vector pPIC9K. Then, the transformants were double-screened on plates containing 0.25 mg/mL and 1.5 mg/mL of neomycin G418 and many clones with different levels of G418-resistance were selected for further studies on expression. After induction by the addition of methanol, various levels of hS83 were detected in the supernatant of P. pastoris containing pPIC9K-hS83. Clones with low G418-resistance produced more hS83 than those with higher G418-resistance. Under the optimized conditions (initial inoculum, 40 A_600nm AU/mL; pH 6.0; methanol concentration, 3.0%; induction time, 72 h), approximately 16–20 mg protein could be recovered from 1 L of the culture. The purified hS83 had a stronger binding ability towards CTLA4-positive Raji cells than CTLA4-negative ECV304 cells. This finding indicates that the antibody produced by P. pastoris is functional and may be used in immunotherapy for cancer, infection, transplant rejection, and autoimmune diseases. Huawei Cai and Lihong Chen contributed equally to this work.     

Recent advances on the GAP promoter derived expression system of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review. Supported by the National High-tech R&D Program (863 program) (No.2007AA021307).     

Bovine gamma-interferon nuclear localization sequence provides translocation of recombinant protein to yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell nucleus

It is known that the expression of heterologous protein production in microorganisms has a negative influence on the host cell. Therefore, to utilize microorganisms for production of recombinant proteins it is necessary the follow the fate of recombinant proteins in cells. In this study, we constructed a modified bovine IFNG gene that encodes interferon with ten amino-acid deletions at the C-terminal. We also generated genetic constructs that ensured the expression of native and modified bovine IFNG fused with GFP gene in yeast Pichia pastoris. The expression of IFN-γ/GFP and IFN-γ(Δ10)/GFP chimeric proteins showed that bovine IFN-γ nuclear localization signal was functioned in yeast cells. The absence of these proteins leads to the cytoplasmic accumulation of recombinant protein.     

Role of N-terminal 28-amino-acid region of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lipase in directing proteins to secretory pathway of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.