Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25–50% less active (p<0.05) at pH 2 to 3.5 or at 45 and 55 °C, but 50% more active (p<0.05) at 75 °C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p<0.05) than that of the glycosylated phytase at 45 and 55 °C, but was 80 and 94% lower (p<0.05) at 65 ° and 75 °C, respectively.     

Expression line approach to recombinant human epidermal growth factor into the yeast,Pichia pastoris from Huh-7 cell line

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.     

High-level expression of non-glycosylated and active staphylokinase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.     

Cloning and Expression of a Clamworm Antimicrobial Peptide Perinerin in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Perinerin, an antimicrobial peptide (GenBank No. P84117), was isolated and characterized from Asian marine clamworms, Perinereis aibuhitensis Grube. This peptide has effects against both gram-positive and gram-negative bacteria in vitro, especially on Pseudemonas aeruginosa. In our study, bioactive perinerin was expressed in Pichia pastoris, and characterized its physicochemical properties. The expressed sample was firstly analyzed by Tricine-SDS-PAGE, after then the recombinant proteins were purified by 2 kD MW cut-off (MWCO) ultrafiltration membrane, and finally purified by 10 kD MWCO ultrafiltration membrane and CM 52-ion exchange chromatography. About 6% protein was obtained by this so called three-purification method. Our results showed that Pichia pastoris was a suitable system for secreting perinerin. Bioactivity assay proved that the recombinant perinerin had antimicrobial effects.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

Glycosylated salivary α-amylases are capable of maltotriose hydrolysis and glucose formation

The physiological and/or clinical significance of sugar chains in human salivary αamylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated α-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G₃) on starch hydrolysis of the amylase fraction, suggests that starch and G₃ can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G₃ hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G₃, G₄, G₅ and G₇, as substrates, the substrate-specificities and G₃/G₅ ratio of amylase activities in the four fractions were examined. Maltopentaose, G₅, is routinely used as a substrate for α-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G₅. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G₃ hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G₄, G₅ and G₇. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.     

Production of bioactive extracellular domain of pig and chicken activin type IIB receptors in Pichia pastoris

Myostatin (MSTN) is a potent negative regulator for skeletal muscle growth, and binds to activin type IIB receptor (ActRIIB) for its cellular signal transduction. Administration of the extracellular domain of ActRIIB (ActRIIB-ECD) improved skeletal muscle growth in laboratory animals, suggesting that ActRIIB-ECD can be a useful pharmacological agent to improve skeletal muscle growth of meat-producing animals. In the current study, pig and chicken ActRIIB-ECDs were produced in the Pichia pastoris GS115, and the recombinant proteins were purified from induced culture media by Ni-NTA affinity chromatography. The digestion of pig and chicken ActRIIB-ECDs with PNGase F and glycoprotein staining demonstrated an N-linked glycosylation of these recombinant proteins. Glycoprotein staining also indicated an additional presence of glycosylation in chicken ActRIIB-ECD. Both the pig and chicken ActRIIB-ECDs were shown to inhibit MSTN activity in a reporter gene assay system in vitro. When MSTN-inhibitory potencies were compared by analyzing EC₅₀ values, no difference in MSTN-inhibitory potency was observed between the glycosylated and N-deglycosylated forms of pig or chicken ActRIIB-ECD, suggesting that glycosylation does not affect the bioactivity of ActRIIB-ECD. MSTN-inhibitory potency of chicken ActRIIB-ECD was greater (P < 0.01) than that of pig ActRIIB-ECD. Results of this study demonstrate that bioactive pig and chicken ActRIIB-ECDs can be produced from P. pastoris. In addition, the study indicates that the N-glycosylation status of ActRIIB-ECD does not affect its bioactivity in vitro.     

Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation

To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.     

Glycosylated salivary alpha-amylases are capable of maltotriose hydrolysis and glucose formation

The physiological and/or clinical significance of sugar chains in human salivary alpha-amylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated alpha-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G3) on starch hydrolysis of the amylase fraction, suggests that starch and G3 can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G3 hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G3, G4, G5 and G7, as substrates, the substrate-specificities and G3/G5 ratio of amylase activities in the four fractions were examined. Maltopentaose, G5, is routinely used as a substrate for alpha-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G5. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G3 hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G4, G5 and G7. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.     

A Family 2a Carbohydrate-Binding Module Suitable as an Affinity Tag for Proteins Produced in Pichia pastoris

The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

A System for Dual Protein Expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

Gibberellin A36, Isolation from Gibberella fujikuroi,Structure and Conversion to Gibberellin A37

A cDNA encoding a putative extracellular α-L-arabinofuranosidase was cloned from the basidiomycete Coprinopsis cinerea (CcAbf62A). CcAbf62A belongs to glycoside hydrolase family 62 (GH62), but is phylogenetically distinct from previously characterized GH62 enzymes. The recombinant CcAbf62A, expressed in Pichia pastoris, released L-arabinose from both wheat arabinoxylan and oat-spelt xylan. The enzyme activity for wheat arabinoxylan was increased by the addition of CcEst1, a carbohydrate esterase from C. cinerea.     

The role of N-glycosylation sites in the activity,stability, and expression of the recombinant elastase expressed by Pichia pastoris

The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70 °C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

With nearly three decades of development, Pichia pastoris (P. pastoris) has become a powerful eukaryotic protein expression system for the expression of thousands of proteins both on a laboratory and industrial scale. In addition, it has also been extensively used as a cell factory for the production of a variety of chemicals. This review summarizes the bottlenecks and solutions in achieving high‐level secretory protein expression with P. pastoris and then outlines its applications on chemical production with an emphasis on its role as whole‐cell biocatalyst. Furthermore, current challenges and future directions of this important system are also discussed.     

Expression in Pichia pastoris and immunological evaluation of a truncated dengue envelope protein

Among the Dengue virus structural proteins, the Envelope glycoprotein is the most important because of its antigenic characteristics. In this work, the E protein from Dengue-2 virus truncated at the C-terminus region was successfully expressed in Pichia pastoris. The E2trunc gene was cloned under the AOX1 promoter from P. pastoris and the signal peptide of the sucrose invertase gene from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression revealed the presence of a protein with the expected size, which was completely associated to the insoluble fraction after cellular disruption. The recombinant N-glycosylated protein reacted with two conformational antibodies against Dengue-2, indicating a proper folding of it. In addition, it was able to induce antiviral antibodies after mice immunization.     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。