Expression and purification of a mutant of human interleukin-2 in Pichia pastoris

Interleukin (IL)-2 is a pharmacologically important cytokine secreted by T-lymphocytes. Recombinant IL-2 (rIL-2) has been modified and produced in many systems. Mass production of rIL-2 is the prerequisite for its wide application. Using a site-directed mutagenesis strategy, we first generated a gene coding for a new type of mutant of human IL-2 (MhIL-2), in which we replaced the cysteine-125 in human IL-2 with alanine, the leucine-18 with methionine, and the leucine-19 with serine. Then we investigated the possibility of its production of MhIL-2 in a Pichia pastoris system. High-level secreted expression of MhIL-2 was achieved by methanol induction. When purified with ultrafiltration, cation-exchange chromatography, and Sephadex G100 gel filtration, about 100 mg of MhIL-2 with high purity was obtained from 1 L of ferment supernatant. Biologic activity assay revealed that the purified recombinant protein displayed increased activity on proliferation of IL-2-dependent CTLL-2 cells. These results suggest that MhIL-2 is an improved IL-2 mutant that might hold great promise for clinical use, and that P. pastoris is an excellent system for the mass production of biologically active hIL-2.     

Constitutive Expression of a rhIL-2-HSA Fusion Protein in Pichia pastoris Using Glucose as Carbon Source

A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040?×?10⁶ IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.     

Expression,Purification, and Characterization of Hepatitis B Virus Surface Antigens (HBsAg) in Yeast <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rushi Liu and Qinlu Lin contributed equally to this work.     

Expression and Production of Human Interleukin-7 in Insect Cells Using Baculovirus Expression Vector System (BEVS)

Interleukin-7 (IL-7) is a glycoprotein cytokine with significant clinical and biomedical potential, such as cancer therapy and HIV infections. Earlier it has been cloned and expressed in various protein expression systems; however, they are not efficient for large-scale production. To address this inadequacy, we report in this paper the production of recombinant human interleukin-7 (hIL-7) in insect cells. A recombinant bacmid containing hIL-7 was constructed, purified, and characterized. It was used to infect Trichoplusia ni (BT1-TN-5B1/High Five™) insect cells. Result shows that T. ni cells successfully produce hIL-7 in shake flask cultures. A scale up to 2.5-L laboratory batch bioreactor showed the efficacy of this system for large-scale production. Our results offer a highly efficient, inexpensive, and convenient system for the large-scale expression and production of recombinant hIL-7.     

Phage presentation and affinity selection of a deletion mutant of human lnterleukin-3

A deletion derivative of the cytokine human interleukin-3 (hIL-3^15–125, comprising amino acids 15–125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble cytokine, phage-presented hIL-3^15–125 exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3^15–125 species (designated hIL-3^15–125 ε) had 20 amino acids appended to its C-terminus; hIL-3^15–125 ε did not exhibit reduced bioactivity. hIL-3^15–125-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

PCR-based Gene Synthesis,Molecular Cloning,High Level Expression,Purification, and Characterization of Novel Antimicrobial Peptide,Brevinin-2R,in <Emphasis Type="Italic">Escherichia Coli</Emphasis>

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.     

Constitutive Expression and Optimization of Nutrients for Streptokinase Production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using Statistical Methods

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett–Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.     

Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Different carrier molecules have been fused to antimicrobial polypeptides (AMPs) to facilitate recombinant protein expression and purification. Some of them have improved the stability of AMPs and reduced the toxicity to host cells, but most current strategies still have some problems to be solved such as poor yield, low purity, high expense, time-consumption, and difficulty in scaling-up. Here, we introduced the elastin-like polypeptides (ELPs) as a fusion partner to express an antimicrobial polypeptide halocidin18 (Hal18). By the reversible soluble–insoluble phase transition, 69 mg of the fusion protein were purified from 1 l of culture medium with the purity of nearly 95%. After cleavage with hydroxylamine, the ELP’s tag was easily separated from Hal18 in the next round of inverse transition cycle and Hal18 (1.7 mg, ∼1.9 kDa) was mainly found in the supernatant with a recovery of about 47% and purity of 60%. Antimicrobial activity showed that Hal18 had strong antimicrobial activity against Escherichia coli and Micrococcus luteus but weak activity against Pichia pastoris.     

Constitutive Expression of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase LIP2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.     

Statistical Determination of Optimal Baculovirus Infection Condition for Recombinant Protein Production in <Emphasis Type="Italic">Drosophila</Emphasis> S2 Cells

Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was >15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.     

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS–PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.     

Induction of the PAOX1-Promoter and Synthesis of GFP Protein in Wild X-33 Strain Recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> Yeast Cells

Recombinant clones of X-33 strain Pichia pastoris containing the marker gene yEGFP were prepared. The optimal methanol concentration in the medium for induction of heterologous expression was determined in the recombinant clones.__________Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 60–62, January–February, 2005.     

Expression and Purification of ZNF191（243-368） in Three Expression Systems

ZNF191 （243-368）, a new human zinc finger protein, probably relates to some hereditary diseases and cancers, To obtain adequate amount of ZNF191（243-368） for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase （GST）, and hexahistidine （6 × His） were used and compared. Among these systems, the expression level of ZNFI91（243-368） was increased in inclusion body system under a higher isopropylthio-β-D-galactoside （IPTG） concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.     

The Effect of Albumin Fusion Patterns on the Production and Bioactivity of the Somatostatin-14 Fusion Protein in Pichia pastoris

Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)₂-human serum albumin (HSA), (SS14)₃-HSA, and HSA-(SS14)₃, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)₃-HSA and HSA-(SS14)₃ was much lower than (SS14)₂-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)₂-HSA being the most effective one. On the whole, (SS14)₂-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity.     

Expression of Recombinant Proteins in <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Cloning and Expression of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 26-2 Lipase Gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and Characterizing for Transesterification

Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut^S and Mut⁺ phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.     

Substrate specificity of <Emphasis Type="Italic">Streptomyces</Emphasis> transglutaminases

Transglutaminase (TGase) is a multifunctional enzyme vital for many physiologic processes, such as cell differentiation, tissue regeneration, and plant pathogenicity. The acyl transfer function of the enzyme can activate primary amines and, consequently, attach them onto a peptidyl glutamine, a reaction important for various in vivo and in vitro protein crosslinking and modification processes. To understand better the structure-function relationship of the enzyme and to develop it further as an industrial biocatalyst, we studied TGase secreted by several Streptomyces species and Phytophthora cactorum. We purified the enzyme from S. lydicus, S. platensis, S. nigrescens, S. cinnamoneus, and S. hachijoensis. The pH and temperature profiles of S. lydicus, S. platensis, and S. nigrescens TGases were determined. The specificity of S. lydicus TGase toward its acyl-accepting amine substrates was characterized. Correlation of the electronic and steric features of the substrates with their reactivity supported the mechanism previously proposed for Streptomyces mobaraensis TGase.     

Expression of Cu,Zn-superoxide dismutase gene from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its resistance to oxidative stress

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.