High level production of stable human serum albumin in Pichia pastoris and characterization of the recombinant product.

Human serum albumin (HSA) is an important therapeutic used in clinical settings for restoration of blood volume and treatment of chemotherapy induced neutropenia. Currently sourced from human serum, it carries the risk of contamination with viruses. The production of stable extracellular recombinant (r)HSA was achieved at nearly 1 g/L at shake-flask level in Pichia pastoris (syn. Komagataella phaffii) containing a three-copy containing HSA expression cassette, prepared in vitro. The HSA specific transcripts were increased by 1.82- to 2.46-fold in the three-copy containing clones indicating increased transcript levels to result in enhanced production of extracellular rHSA. The purified rHSA displayed secondary structure, zeta potential, size distribution and biological efficacy that matched with that of the commercial HSA. Cultivation strategy was developed at bioreactor level for the single HSA expression cassette containing recombinant which led to productivity of 300 mg/L/d of rHSA with minimum proteolytic cleavage.

Development of a dual specific growth rate-based fed-batch process for production of recombinant human granulocyte colony-stimulating factor in Pichia pastoris.

A number of limitations exist for production of human granulocyte colony-stimulating factor (G-CSF) in Pichia pastoris. In this study, two different specific growth rates (0.015 h-1, 0.01 h-1) were used sequentially in the mixed substrate feeding period during methanol induction phase to enhance the G-CSF titer in the culture broth. Necessary parameters required for implementing the feeding strategy, such as specific product yield on biomass (YP/X) and maintenance coefficient (m) on glycerol, methanol, and sorbitol were estimated using continuous culture technique. Using this strategy, for the same volumetric productivity, about 20% increase in protein titer was achieved over that obtained from the run carried out at a single pre-set value of 0.015 h-1 alone. Thus, implementation of higher specific growth rate (0.015 h-1) set during initial stages of the methanol induction phase followed by a lower specific growth rate (0.01 h-1) helped in achieving increased protein titers.

Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

Production of cellobiose dehydrogenase from a newly isolated white rot fungus Termitomyces sp. OE147.

Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box-Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.