Expression, purification, and immunogenic characterization of Epstein–Barr virus recombinant EBNA1 protein in Pichia pastoris

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4⁺ and CD8⁺ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.     

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains

Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)₆ tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.     

Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expressing protein disulfide isomerase

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Protein folding and disulfide bond formation were identified as rate-limited steps in recombinant protein secretion in yeast cells. For the production of β-glucosidase in Pichia pastoris, final β-glucosidase activity reached 1,749 U/mL after fermentation optimization in a 3 L bioreactor, while the specific activity decreased from 620 to 467 U/mg, indicating a potential protein misfolding. To solve this problem, protein disulfide isomerase, a chaperone protein which may effectively regulate disulfide bond formation and protein folding, was co-expressed with β-glucosidase. In the co-expression system, a β-glucosidase production level of 2,553 U/mL was achieved and the specific activity of the enzyme reached 721 U/mg, which is 1.54 fold that of the control.     

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris

Human α-defensin 5 (HD₅), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD₅ through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD₅ mature peptide (rmHD₅) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD₅ by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD₅ into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD₅ vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD₅ nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD₅, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD₅. The results showed that about 165.0 mg/l of rmHD₅ with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD₅ was recovered after purification. The in vitro experiments revealed that rmHD₅ exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD₅ in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.     

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.     

Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris

Pichia pastoris is widely used for the production of recombinant proteins in industrial biotechnology. In general, industrial production processes describe fed-batch processes based on the specific growth rate. Recently, we introduced the specific substrate uptake rate (q _s) as a novel parameter to design fed-batch strategies for P. pastoris. We showed that a dynamic feeding strategy where the feed was adjusted in steps to the maximum specific substrate uptake rate was superior to more traditional strategies in terms of specific productivity. In the present study, we compare three different dynamic feeding strategies based on q _s for a recombinant P. pastoris strain with respect to cell physiology, methanol accumulation, productivity and product quality. By comparing (A) a feeding profile at constant high q _s, (B) a periodically adjusted feeding profile for a stepwise q _s ramp, and (C) a feeding profile at linear increasing q _s, we evaluated potential effects of the mode of feeding. Although a dynamic feeding strategy with stepwise increases of q _s to q _{s max} resulted in the highest specific productivity, a feeding profile where the feeding rate was stepwise increased to a constant high q _s value was superior in terms of the amount of active enzyme produced and in the amount of accumulated methanol. Furthermore, this feeding strategy could be run automatically by integrating an online calculator tool, thus rendering manual interventions by the operator unnecessary.     

Elimination of β-mannose glycan structures in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.     

Validation of a new restraint docking method for solution structure determinations of protein–ligand complexes

A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein–ligand, ligand–ligand, and protein–protein restraints for residues in or near to the binding site, combined with a set of protein–protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in ¹H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase.trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase.methotrexate reference structure.     

Production of Aspergillus niger β-mannosidase in Pichia pastoris

β-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various sugar chains. cDNA corresponding to the β-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The β-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44mmol/l for p-nitrophenyl β-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).     

Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59–97 above kDa glycoprotein, 52–57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.     

Purification and characterization of a β-1,3-glucomannanase expressed in Pichia pastoris

The glycoside hydrolase β-1,3-glucomannanase is an enzyme that specifically breaks the β-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0 mg/l and 224.0 mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 °C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50 °C and 4.5, respectively. The recombinant plMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of β-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas.     

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris

Bioprocess and Biosystems Engineering - Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing....     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

Surface histidine mutations for the metal affinity purification of a β-carbonic anhydrase

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.     

Expression of the gene coding for bacterial hemoglobin improves β-galactosidase production in a recombinant Pichia pastoris

Genes coding for Vitreoscilla hemoglobin (VHb) with peroxisome targeting signal (PTS1) tag and -galactosidase were co-expressed in Pichia pastoris under the alcohol oxidase1 (AOX1) promoter. The expression of VHb-PTS1 had no positive effect on cell growth but significantly enhanced the whole cell -galactosidase activity to 4-fold higher than that of VHb-free cell in yeast extract/peptone/methanol medium under aerobic cultivation.     

Production of bioactive sheep β-defensin-1 in Pichia pastoris

Previous research has shown that sheep β-defensin-1 (sBD-1), a small cationic peptide with a broad range of antimicrobial activities, could inhibit the growth of both Gram-positive and Gram-negative bacteria as well as that of fungi. In order to increase the yield of current ovine defensin purification methods, mature sBD-1 (msBD-1) was added with a 6-His tag on the C-terminus (msBD-1-T) and expressed in Pichia pastoris in the presented work. The msBD-1 and msBD-1-T were expressed in the Pichia pastoris. Both msBD-1 and msBD-1-T were purification, and the two peptides were used to inhibit Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Shigella flexneri. The antimicrobial activity of the 6-His tagged msBD-1-T peptide was not significantly different from that of the native msBD-1 peptide. The two peptides could inhibit the growth of Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Shigella flexneri with equal efficiency as well as chemoattractant function. In addition, the yield of purified 6-His-tagged msBD-1 was greater than that of msBD-1. The presented method might be a more efficient approach to produce bioactive sBD-1.     

Constitutive expression of alkaline β-mannanase in recombinant Pichia pastoris

Alkaline β-mannanase has important applications for specific industrial processes like pulp bleaching and the detergent industry. The low yield of alkaline β-mannanase produced from native microbes such as alkaliphilic Bacillus limits its applications. Pichia pastoris is the most efficient heterologous system to produce alkaline mannanase. However, the previous use of the AOX system required large amount of methanol and sophisticated operation strategy, which are undesirable in large scale production. In this study, we established a safe and simple constitutive expression process for mannanase production in P. pastoris. The mannanase gene was successfully expressed under the control of GAP promoter. Sequential optimization of the constructed strains was also performed including the copy number optimization and co-expression of chaperone genes. A two-stage feeding strategy was then applied for the finally optimized strain. After 96 h fermentation, a production level of 2980 U/mL was finally reached, illustrating the potential of the GAP constitutive expression system for industrial scale preparation of alkaline β-mannanase.     

Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris

The β-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal β-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.