Navin Khanna

Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies

A strategy is described for improved refolding and purification of recombinant human gamma interferon (rh-IFN gamma), which could warrant a higher yield and specific activity than reported previously. The optimal conditions of refolding are obtained by addition of a labilizing agent, L-arginine, in the refolding buffer. A 10-fold increase in the yield was observed with 0.5 M L-arginine, compared with renaturation in its absence. By varying renaturation parameters, the conditions that allow functional refolding of approximately 25-30% of the recombinant protein have been standardized. A simple process is also described for the purification of rh-IFN gamma. The purification involves a single-column chromatography on S-Sepharose, after refolding of rh-IFN gamma in arginine containing buffer. This procedure has consistently produced rh-IFN gamma having a purity of at least 97%, the rest being the aggregated form of gamma interferon. The purified protein is a dimer under non-denaturing conditions and has a specific activity of 2 x 10(8) IU mg-1 protein, as measured by viral cytopathic assay.

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.

Production of interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins

Escherichia coli TG1 transformed with a temperature-regulated interferon-α expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of ∼4 g interferon-α/l culture broth. Interferon-α was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-α was ∼300 mg/l with respect to the original high cell density culture broth (overall yield of ∼7.5% active interferon-α). The purified recombinant interferon-α was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of ∼2.5 × 108 IU/mg based on viral cytopathic assay.

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

BackgroundHepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus.ResultsLarge-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth.ConclusionIn comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen.

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.

Replication-Defective Adenoviral Vaccine Vector for the Induction of Immune Responses to Dengue Virus Type 2

ABSTRACT A recombinant replication-defective adenovirus vector that can overexpress the ectodomain of the envelope protein of dengue virus type 2 (NGC strain) has been constructed. This virus was immunogenic in mice and elicited dengue virus type 2 specific B- and T-cell responses. Sera from immunized mice contained neutralizing antibodies that could specifically recognize dengue virus type 2 and neutralize its infectivity in vitro, indicating that this approach has the potential to confer protective immunity. In vitro stimulation of splenocytes (from immunized mice) with dengue virus type 2 resulted in a significant proliferative response accompanied by the production of high levels of gamma interferon but did not show significant changes in interleukin-4 levels. This is suggestive of a Th1-like response (considered to be important in the maturation of cytotoxic T lymphocytes that are essential for the elimination of virus-infected cells). The data show that adenovirus vectors offer a promising alternative strategy for the development of dengue virus vaccines.

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

BackgroundThe prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.ResultsA synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.ConclusionsA simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity

Dengue is a mosquito-borne viral disease caused by four antigenically distinct serotypes of dengue viruses (DENVs). This disease, which is prevalent in over a hundred tropical and sub-tropical countries of the world, represents a significant global public health problem. A tetravalent dengue vaccine capable of protecting against all four DENV serotypes has been elusive so far. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we have utilized a discrete carboxy-terminal region of the major DENV envelope (E) protein, known as domain III (EDIII), which mediates virus entry into target cells and contains multiple serotype-specific neutralizing epitopes, to create a chimeric tetravalent antigen. This antigen derived by in-frame fusion of the EDIII-encoding sequences of the four DENV serotypes was expressed using a replication-defective recombinant human adenovirus type 5 (rAdV5) vaccine vector. This rAdV5 vector induced cell-mediated immune responses and virus-neutralizing antibodies specific to each of the four DENVs in mice. Interestingly, anti-AdV5 antibodies did not suppress the induction of DENV-specific neutralizing antibodies. We observed that anti-AdV5 antibodies in the sera of immunized mice could promote uptake of a rAdV5-derived reporter vector into U937 cells, suggesting that pre-existing immunity to AdV5 may in fact facilitate the uptake of rAdV5 vectored vaccines into antigen presenting cells. This work presents an alternative approach to developing a single component tetravalent vaccine that bypasses the complexities inherent in the currently adopted four-in-one physical mixture approach.

Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the hepatitis B surface antigen.

BackgroundA protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.ResultsHigh level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.ConclusionsIt is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.