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.

BACKGROUND: The 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. RESULTS: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae alpha-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 approximately 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 approximately 1.5 g of 99% pure recombinant human insulin per liter of culture broth. CONCLUSIONS: A 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.

Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv.

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1 M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precursor revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation.

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.