Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

• Published in: Applied biochemistry and biotechnology 2013-09, Vol.171 (1), p.10-19
• Main Authors: A.R., Sudheer Reddy, Satheeshkumar, Padikara Kutty, Vijayalakshmi, Mookambeswaran A.
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Coagulation; Culture media; Factor VIII - chemistry; Factor VIII - genetics; Factor VIII - isolation & purification; Factor VIII - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Engineering - economics; Genetic Engineering - methods; Genetic Vectors - genetics; Hemophilia; Human factors; Humans; Lipids; Phospholipids - metabolism; Pichia - genetics; Pichia pastoris; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Transformation, Genetic; Yeasts
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-013-0338-4

Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1–A2) and light chains (A3–C1–C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.