Pichia Pastoris Expression System Research Articles

Expanding the Genetic Repertoire of the Methylotrophic Yeast Pichia pastoris

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.

High-Yield Expression and Purification of a Plasma Membrane Citrate Transport Protein

Citrate is prominently and uniquely involved in metabolic crossroads that govern the energy balance of human and other organisms, yet little is known about the molecular mechanisms determining its entry into cells. The unique ability of plasma membrane citrate transporters (PMCTs) to import extracellular citrate into the cytoplasm confers upon them an essential role in determining an organism's energy state. Thus, PMCTs located in liver and brain figure prominently in: i) fatty acid, lipid, and cholesterol biosyntheses; ii) control of glucose metabolism via the citrate-mediated allosteric inhibition of PFK-1; and iii) neuronal cell synthesis of neurotransmitters. Therefore, inhibition of human PMCT function may prove to be a novel strategy for altering energy balance in a manner that mimics caloric restriction. With this background in mind, we recently cloned PMCTs from C. elegans, Drosophila, mouse, rat, and human into the Pichia pastoris expression system and developed a biofermentation protocol for high-yield expression of the human and the C. elegans PMCTs. We have been able to successfully purify detergent-solubilized C. elegans PMCT via sequential chromatography on Ni-NTA, Talon, and Superdex, resulting in material that is > 90% pure. Mass spectrometry confirms that full-length C. elegans PMCT was the purified entity. Preliminary studies indicate that we have been able to successfully reconstitute detergent-solubilized C. elegans PMCT function in liposomal vesicles. Sulfhydryl reagents such as NEM and pCMB effectively inhibit the reconstituted transporter. These studies provide the foundation for a detailed structure/function analysis of this metabolically essential plasma membrane transport protein. Supported by NIH grant GM-054642 to R.S.K.

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (? and ?) of the enzyme were N-glycosylated, while the ?-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view.

Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from Ganoderma lucidum in Pichia pastoris

The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in anti-tumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis to a degree of about 32.4%. Taken together, the FIP gene from Changbai G. lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l−1). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies.

Paclitaxel Binding to Human and Murine MD-2

Paclitaxel (PTX) is an important cancer chemotherapeutic agent that binds to beta-tubulin and prevents mitosis through microtubule overstabilization. Recent evidence also implicates PTX in the induction of apoptosis of cancer cells via the TLR4 innate immune pathway. The TLR4 accessory protein, MD-2, is an essential component for the species-specific proinflammatory activity of PTX on murine cells. However, whether PTX binds to human MD-2 and how MD-2 and TLR4 interact with PTX are not well defined. Recombinant human MD-2 (rhMD-2) was produced in a Pichia pastoris expression system, and the interaction between rhMD-2 and PTX was assessed by an enzyme-linked immunosorbent assay to show that PTX binds rhMD-2. Formation of the latter complex was found to be dose-dependent and inhibited by anti-MD-2 antibody but not by an isotype control antibody. As measured by human tumor necrosis factor alpha production, human THP-1 monocytes expressing TLR4 and MD-2 were poorly responsive to the addition of PTX, but murine macrophages expressing TLR4 and MD-2 responded in a dose-dependent manner. Human embryonic kidney (HEK293) cells transfected with both human TLR4 and human MD-2 or human MD-2 and murine TLR4 were also poorly responsive to PTX (10 microm). However, HEK293 cells transfected with murine MD-2 and human TLR4 or murine MD-2 and murine TLR4 were highly responsive to PTX (10 microm), indicating that the murine MD-2/PTX interaction is required for TLR4 activation. To further define the structural differences for MD-2/TLR4 activation, crystal structures of both murine and human MD-2 were subjected to PTX docking by computational methods. These models indicate that PTX binds in the pocket of both human and mouse MD-2 structures. The species-specific difference between human and murine MD-2 activation of TLR4 by PTX can be explained by alterations of surface charge distribution (i.e. electrostatic potential), binding pocket size, and the locus of PTX binding within the MD-2 pocket, which results in reorganization of the 123-130 amino acid loop. In particular, Phe(126) appears to operate as a bridge for TLR4.MD-2 dimerization in the mouse but not the human protein.

Recent advances on the GAP promoter derived expression system of Pichia pastoris

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.

Expression, purification, and functional characterization of recombinant human interleukin-7

Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7’s effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler purification steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple purification strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the purified rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a specific activity comparable to commercial sources. These results suggest that purification of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL-7 for clinical applications as well as basic researches.

Toxoplasma gondii: Serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti- Toxoplasma human serum, pooled anti- Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients ( n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites ( P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti- Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.

Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZalphaA vector under the control of AOX1 promoter, which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native N- and C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Characterization and cloning of chitin deacetylases from Rhizopus circinans

Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of ∼75kDa. At 37°C it shows optimal activity at pH 5.5–6. Its pH stability and thermal stability are good. Mn2+ and Mg2+ slightly enhance the activity of the enzyme and Cu2+ strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of ∼75kDa and, like the native enzyme, showed optimal activity at pH 5.5–6 at 37°C. It was strongly inhibited by Cu2+. The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.

Mutation of human plasminogen kringle 1–5 enhances antiangiogenic action via increased interaction with integrin αvβ3

Angiogenesis plays a primary role in tumor growth and metastasis. Angiostatin, a proteolytic fragment containing the first four kringle domains of human plasminogen, can inhibit angiogenesis. The anti-angiogenic activities of kringle 1-5 (K(1-5)) and kringle 5 fragments of plasminogen are greater than angiostatin in inhibiting angiogenesis and angiogenesis-dependent tumor growth. To further optimize kringle fragment anti-angiogenic activities, mutations were created at the potential glycosylation sites Asn-289 and Thr-346 and the Lys binding site, Leu-532, at kringle 5, including K(1-5)N289A (replacing Asn by Ala at residue 289), K(1-5)T346A, K(1-5)L532R, K(1-5)N289A/T346A, K(1-5)T346A/L532R, K(1-5)N289A/L532R, and K(1-5)N289A/T346A/L532R. Wild-type and mutant K(1-5) proteins were expressed successfully by the Pichia pastoris expression system. Native K(1-5) from proteolytic cleavage and wild-type K(1-5) have similar activity in inhibiting basic fibroblast growth factor-induced endothelial cell proliferation. Among these mutated proteins, K(1-5)N289A/T346A/L532R exhibited the greatest effect in inhibiting endothelial cell proliferation and in inducing endothelial cell apoptosis. Integrin alpha(v)beta(3)-mediated adhesion of K(1-5)N289A/T346A/L532R to endothelial cells was more greatly enhanced when compared to wild type K(1-5). Furthermore, K(1-5)N289A/T346A/L532R was most potent in inhibiting basic fibroblast growth factor-induced angiogenesis in Matrigel assay in vivo. Angiogenesis-dependent tumor growth was inhibited by systemically injected K(1-5)N289A/T346A/L532R into mice. These results demonstrate that alteration of glycosylation and Lys binding properties could increase the anti-angiogenic action of K(1-5), possibly via enhanced interaction with integrin alpha(v)beta(3) in endothelial cells.

Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris 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.

6-S-Cysteinylation of Bi-covalently Attached FAD in Berberine Bridge Enzyme Tunes the Redox Potential for Optimal Activity

A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to approximately 6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of +132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of +53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-alpha position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor.

A novel murine protein with no effect on iron homoeostasis is homologous with transferrin and is the putative inhibitor of carbonic anhydrase

In a search for genes that modify iron homoeostasis, a gene (1300017J02Rik) was located immediately upstream of the murine TF (transferrin) gene. However, expression of the 1300017J02Rik gene product was not responsive to a number of modulators of iron metabolism. Specifically, expression was not altered in mouse models of iron disorders including mice with deficiencies in the haemochromatosis protein Hfe, the recombination-activating protein, Rag, beta2-microglobulin, TF, ceruloplasmin or Hb, or in mice with microcytic anaemia. Additionally, neither lipopolysaccharide nor hypoxia treatment resulted in any significant changes in the 1300017J02Rik expression level. The genomic DNA sequence suggested that the 1300017J02Rik gene product might be a protein equivalent to the pICA {porcine ICA [inhibitor of CA (carbonic anhydrase)]}. The coding region for the murine 1300017J02Rik gene was placed into the pNUT expression vector. Transformed BHK cells (baby-hamster kidney cells) were transfected with this plasmid, resulting in secretion of recombinant mICA (murine ICA) into the tissue culture medium. Following purification to homogeneity, the yield of mICA from the BHK cells was found to be considerably greater (at least 4-fold) than the yield of pICA from a previously reported Pichia pastoris (yeast) expression system. MS showed that the recombinant mICA was a glycoprotein that associated with CA in a 1:1 stoichiometry. Despite its high sequence similarity to TF, titration experiments showed that mICA was unable to bind iron specifically. Although enzymatic assays revealed that mICA was able to inhibit CA, it is unclear if this is its sole or even its major function since, to date, humans and other primates appear to lack functional ICA. Lastly, we note that this member of the TF superfamily is a relatively recent addition resulting from a tandem duplication event.

Saposin B mobilizes lipids from cholesterol‐poor and bis(monoacylglycero)phosphate‐rich membranes at acidic pH

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A-D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid-binding and -transfer proteins show different specificities and varying modes of action. Water-soluble SAPs facilitate the degradation of membrane-bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane-water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap-B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap-B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap-B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap-B is essential to achieve its full lipid-extraction activity. Removal of the carbohydrate moiety of Sap-B reduces its membrane-destabilizing quality. An unglycosylated Sap-B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.

Expression, purification, and activity identification of Alfimeprase in Pichia pastoris

Alfimeprase (ALF) is a truncated form of non-hemorrhagic zinc metalloproteinase fibrolase. In order to achieve a high level secretion and full activity expression of ALF, the Pichia pastoris ( P. pastoris) expression system was used. ALF coding sequence fused with a 6∗histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector pPIC9K and then expressed in P. pastoris strains of GS115 and KM71 by methanol induction. SDS–PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to mouse anti-His· tag monoclonal antibody. Under the optimized culture parameters of pH value, initial A 600 value, methanol daily addition concentration and induction time length, the production of rALF reached up to 510 mg/L and 465 mg/L of the GS115 and KM71 transformants, respectively. It also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purity of rALF was as high as 96%. The fibrinolytic activity of rALF revealed by the modified fibrin plate method indicated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. The improved expression system will facilitate further studies and industrial production of ALF.

Cloning, expression, purification and functional characterization of recombinant human PSP94

Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS–PAGE analysis and a peak with a molecular mass ( M r) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, i n vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein(HPV16 L1) in Pichia pastoris

In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZαB. HPV16 L1 protein expression induced by methanol was screened by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The results indicate that the HPV16 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into virus-like particles(VLPs), which show a good immunogenicity and induces high-titer antibody in mice.

Expression, purification and characterization of human IFN-λ1 in Pichia pastoris

Interferon-lambda (IFN-lambda) is a newly identified IFN family which belongs to the class II cytokines. The three members of this family represent antiviral activities like other IFNs. In the present study, recombinant human IFN-lambda1 (rhIFN-lambda1) was produced by using the methylotrophic yeast Pichia pastoris (P. pastoris) expression system. cDNAs encoding amino acids 23-200 or 20-200 of human IFN-lambda1 were cloned and joined to sequence encoding the leader region (prepro segment) of the precursor of Saccharomyces cerevisiae alpha-factor. The two hybrid genes were subcloned into yeast integrative vector pAO815 separately to construct expression plasmids bearing four tandem copies of IFN-lambda1 expression cassettes. The expression plasmids were then used to transform into P. pastoris strain GS115, resulting in recombinant strains GS115/IFNlambda1P and GS115/IFNlambda1G with Mut(+) or Mut(s) phenotype. rhIFN-lambda1 was secreted into the medium upon methanol induction. In GS115/IFNlambda1P, however, KEX2 cleavage for mature rhIFN-lambda1 generation was inhibited by a proline at [Formula: see text] and the products were different from anticipation. GS115/IFNlambda1G strain secreted two forms of mature rhIFN-lambda1 with the same N-terminal sequence and different molecular weight. Periodic acid-Schiff (PAS) staining indicated that these proteins were glycosylated. The yield of low-glycosylated rhIFN-lambda1 in GS115/IFNlambda1G strain was approximately 65 mg l(-1) in shaking flasks, representing around 57% of the total secreted proteins. rhIFN-lambda1 was purified by cation exchange chromatography and gel filtration. The purified rhIFN-lambda1 showed specific efficiency to activate signal transducer and activator of transcription 1 (STAT1) and STAT2 that was comparable to that of commercial IFNalpha2a.

Effect of cAMP-dependent protein kinase A (PKA) on HCV nucleocapsid assembly and degradation

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.