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.     

Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris

The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml^?1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml^?1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation.     

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.     

Synthesis of biologically active human interferon α-2b in Nicotiana benthamiana

A method was developed for the production and purification of biologically active recombinant human interferon α-2b (rhIFN α-2b) synthesized by expression in Nicotiana benthamiana plants. A gene construct containing a modified hIFN α-2b gene was cloned in two vectors based on tobacco mosaic virus driven by an actin promoter from Arabidopsis thaliana (pA-IFN-A) and cauliflower mosaic virus driven by a 35S promoter (pA-IFN-S). The expression vectors were introduced into the plant cells by agroinfiltration. The maximum rates of synthesis achieved in the case of pA-IFN-A and pA-IFN-S 5 days after agroinfiltration were determined to be 200 and 20 mg per 1 kg of fresh leaves, respectively. The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.     

Secretory expression of the α-subunit of human coagulation factor XIII in the yeast Pichia pastoris

Pro-FXIIIa (the -subunit of FXIII with activation peptide, which must be removed to produce the active form of FXIIIa), cloned from human placenta cDNA library, was overexpressed in the methylotrophic yeast Pichia pastoris GS115 (his4) and secreted into the culture medium to yield the recombinant pro-FXIIIa subunit with a predicted molecular mass of approximately 83 kDa. The gene was located immediately downstream of the strong yeast alcohol oxidase promoter (AOX1). In shake flask culture, recombinant pro-FXIIIa (rFXIIIa) was secreted into the culture medium at above 50 mg l^–1. The fibrin-stabilizing activity of the recombinant pro-FXIIIa, after thrombin activation, was confirmed using fibrin cross-linking patterns, and analyzed by SDS-PAGE.     

Enhanced production of human FcγRIIa receptor by high cell density cultivation of Escherichia coli

Human Fc receptors (FcγR) are membrane glycoproteins that are expressed on all immunologically active cells and have a well-defined role in regulating innate and adaptive immune responses by binding to the immunoglobulin G (IgG) antibody. Among the several classes of Fc receptors, FcγRIIa is the most widely expressed, and it serves as an important reagent in antibody engineering. Here, we report on high cell density cultivations (HCDC) of Escherichia coli for preparative scale production of FcγRIIa in a 6.6L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD(600) of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD(600) of 100), the cell density increased to an OD(600) of 234 within 9h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD(600) of 46). After simple purification steps including denaturation and refolding, 87.7 mg of soluble FcγRIIa that was more than 95% pure was obtained from a 20-mL culture with high recovery yield (≈54%). The biological activity of purified FcγRIIa was also confirmed by evaluating its interaction with all subclasses of IgG antibodies using an ELISA bioassay.     

Production and crystallization of processing α-glucosidase I: Pichia pastoris expression and a two-step purification toward structural determination

Eukaryotic N-glycoprotein processing in the endoplasmic reticulum begins with the catalytic action of processing α-glucosidase I (αGlu). αGlu trims the terminal glucose from nascent glycoproteins in an inverting-mechanism glycoside hydrolysis reaction. αGlu has been studied in terms of kinetic parameters and potential key residues; however, the active site is unknown. A structural model would yield important insights into the reaction mechanism. A model would also be useful in developing specific therapeutics, as αGlu is a viable drug target against viruses with glycosylated envelope proteins. However, due to lack of a high-yielding overexpression and purification scheme, no eukaryotic structural model of αGlu has been determined. To address this issue, we overexpressed the Saccharomyces cerevisiae soluble αGlu, Cwht1p, in the host Pichia pastoris. It was purified in a simple two-step protocol, with a final yield of 4.2mg Cwht1p per liter of growth culture. To test catalytic activity, we developed a modified synthesis of a tetrasaccharide substrate, Glc(3)ManOMe. Cwht1p with Glc(3)ManOMe shows a K(m) of 1.26 mM. Cwht1p crystals were grown and subjected to X-ray irradiation, giving a complete diffraction dataset to 2.04 ? resolution. Work is ongoing to obtain phases so that we may further understand this fundamental member of the N-glycosylation pathway through the discovery of its molecular structure.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon 2b (IFN2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN2b were analysed by [³H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

This paper studies the biological activity of protein extracts of the Cichorium intybus L. and Lactuca sativa L. transgenic plants with the human interferon ??2b gene againsf vesicular stomatitis virus. Extracts from transgenic lettuce and chicory roots, which were obtained after A. rhizogenes-mediated transformation, had antiviral activity in the range 1620?C5400 IU/g of weight; extracts from leaves of chicory plants transformed by A. tumefaciens, up to 9375 IU/g. The dependence of the antiviral activity of plant extracts from roots or leaves on the vector used for plant transformation is shown. The extracts of plant roots obtained by A. rhizogenes-mediated transformation had antiviral activity; at the same time, such activity was absent in the extracts from leaves.     

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l^?1 sucrose, 0.5 mg l^?1 of the auxin 1-naphthalene acetic acid, and 0.5 mg l^?1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture

Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h⁻¹ for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y _P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l⁻¹ h⁻¹.     

High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris

A novel β-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized β-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular β-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for β-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-β-D-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminarin. The present results should contribute to improving industrial production of β-glucosidase.     

Production of high activity Aspergillus niger BCC4525 β-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis

A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high k_cat/K_m (461 mg^?1 s^?1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant β-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.     

Studies of α-protein in human cell cultures

Summary α-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium A₃. AGF is effective at less than 0.4 μg per ml. By using the procedures described in the text, it is possiblee to culture HeLa cells is very simple media such as Eagle's basal medium. The properties of AGF are such that it may be adsorbed on glass or plastic flasks. Glass flasks treated with AGF retain full activity after washing with acetone, and treatment with ethyl ether and chemically defined medium. Adsorbed AGF is destroyed by trypsin. AGF can detoxify protamines, polylysines or histones. It will reverse the aggregation response induced by adding complexes composed of carboxymethylcellulose (CMC) and basic proteins. The results support the contention that highly adsorptive AGF functions at the cell surface and is capable of modifying the response of the cell to its environment.     

Improved expression of Rhizopus oryzae α-amylase in the methylotrophic yeast Pichia pastoris

In our previous study, the α-amylase from Rhizopus oryzae (RoAmy) was expressed in Escherichia coli and Saccharomyces cerevisiae but the obtained recombinant RoAmy (rRoAmy) yields were too low. The aim of the present research was to obtain high-level expressions of RoAmy in the methylotrophic yeast Pichia pastoris. To this end, we constructed P. pastoris strains with the capability to express recombinant α-amylase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) and methanol-inducible alcohol oxidase 1 promoters. The levels of inducibly expressed rRoAmy were higher than those of constitutively expressed. The maximal inducible rRoAmy expression levels for the Mut(+) strains (41.1mg/l) were approximately eight times higher than those for the Mut(s) strains and 24 times higher than those expressed under the control of the GAP promoter. For both inducible and constitutive expressions, the S. cerevisiae α-prepro sequence and the native signal sequence of RoAmy were used separately to direct the secretion of rRoAmy into the culture medium of P. pastoris. Low levels of intracellular amylase activities that had been detected after shake-flask fermentation indicated that both signal sequences could effectively direct the secretion of rRoAmy under all studied conditions. In addition, the secretion levels of rRoAmy directed with its own signal peptide were 7-10% higher than those directed by the α-prepro sequence.