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.

Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin.

Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110-380 mg L-1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.

Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris.

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α-factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

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.