High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

High-level secretory expression, purification and characterization of Ailuropoda melanoleuca growth hormone in Pichia pastoris

Growth hormone is one of the most important hormones, which is involved in many reproductive processes of giant panda Ailuropoda melanoleuca. In this study, the mature peptide of A. melanoleuca growth hormone (AmGH) was successfully expressed and secreted in Pichia pastoris under the control of AOX1 promoter. The expression condition for AmGH in P. pastoris, such as the expression time, pH value and methanol concentration in the BMMY were optimized and the AmGH expression level is about 100 mg/L using GS115 recombinant under optimized condition (96 h of 1.5% methanol induction). The secreted nascent AmGH were purified using ammonium sulfate fractionation. The mature AmGH protein exhibited a molecular mass of approximately 22 kDa on SDS–PAGE. This study would provide a new opportunity for large-scale expression and purification of AmGH, which might facilitate studies on the biological activity of AmGH.     

High-level expression and purification of recombinant human catalase in Pichia pastoris

Catalase is one of the antioxidant enzymes and is involved in many pathophysiologic processes and human diseases. This study focused on high-level expression and purification of recombinant catalase in Pichia pastoris. The cDNA encoding catalase was cloned by RT-PCR from Fetal liver of Homo sapiens. After PCR and construction of expression vector pPIC9K-CAT, human catalase was expressed highly in P. pastoris yeast SMD1168 and secreted into the culture medium. The secreted catalase was purified to a purity of 95% by ammonium sulfate fractionation, anionic exchange-chromatography, and Macro-prep Ceramic Hydroxyapatite with a overall yield of 60%. This study provides a new method for large-scale expression and purification of recombinant protein catalase.     

High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization

Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS-PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed.     

Expression,characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris

Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene. Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs. In contrast, P. pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form. Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 microM) resulted in significant improvements in expression of rPLF from P. pastoris. Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L). P. pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column. The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF.     

Soluble expression and purification of porcine pepsinogen from Pichia pastoris

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).     

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris

A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca²⁺ and Mg²⁺ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K _m) of 1.89 mg/mL and a maximum reaction rate (V _max) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.     

Expression, purification, and characterization of equistatin in Pichia pastoris

Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina. It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins. Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields. A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P. pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 transformants. Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor. EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI.     

Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration

A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55 °C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited. Moreover, our study showed that a portion of SPTK was N-glycosylated during fermentation. The activity and thermal stability of the recombinant SPTK were improved after the removal of glycosylation, and the N-glycosylation of SPTK could be efficiently removed through co-culture with P. pastoris strains expressing Endo-β-N-acetylglucosaminidase H. We constructed expression vectors harboring from one to four repeats of Sptk-expressing cassettes via an in vitro BioBrick assembly approach. And the result of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the genome of P. pastoris through a single recombination event. These strains were used to study the correlation between the gene copy number and the expression level of SPTK. The results of qPCR and enzyme activity assays indicated that the copy number variation of Sptk gene generally had a positive effect on the expression level of SPTK, while an increase in integration of target gene did not guarantee its high expression. The maximum yield and specific activity of SPTK in P. pastoris were obtained from the recombinant yeast strain harboring two-copy tandem Sptk-expressing cassettes, the yield reached 0.48 g/l after a 6-d induction using menthol in shake flasks and 3.2 g/l in high-density fermentation with specific activity of 5200 U/mg. In addition, the recombinant SPTK could efficiently degrade chicken feather and hydrolyzed the gelatin layer of photographic film. These properties made the recombinant SPTK a suitable candidate for industrial applications and for eliminating the pollution of keratin.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.     

High-level expression of a biologically active staphylokinase in Pichia pastoris

Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310?mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20–37°C and pH ranges of 6.8–9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042?U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720?µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6?kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.     

Expression, purification and characterization of rat angiopoietin-2 in Pichia pastoris

Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.