Expression And Purification Of Recombinant Human Serum Albumin In Hansenula Polymorpha

Human serum albumin (HSA) plays an important role in clinical therapy. At present, HSA prepared from human plasma is limited in supply and there is the potential risk of contamination of the product by blood-drived pathogens. It is of great significance to product recombinant HSA by genetic engineering means.Hansenula polymorpha as well as Pichia pastoris is capable of growing with methanol as its sole carbon and energy source. Based on Hansenula polymorpha's genetic codon usage bias, the optimized gene of HSA was synthesized and cloned into Hansenula polymorpha expression vector pDGXMPT1.0 to construct recombinant HSA (rHSA) expression plasmid. After transformed into Hansenula polymorpha strain by electroporation, the transformants were screened on MD medium for URA3~+ phenotype. The high-expression strain was selected by the methods of SDS-PAGE and Western-blotting. Fed-batch fermentation process was explored in a 30L-fermenter. Highest expression level of 1.033g/L was achieved. at 61-h post inducton, whereafter the proteolytic degradation of rHSA turned obviously. The concentration of rHSA decreased to a low level 150h after induction. The rHSA was purified by streamline SP exchange chromatography, phenyl Bio-sep 6FF hydrophohic interaction chromatography and DEAE sepharose exchange chromatography. Electrophoresis-grade protein was obtained. Diluting the purified protein to approprite concentration, we immunized several group of mice and sera were collected. Double agar diffusion test and ELISA demonstrated that rHSA showed almost identical antibody-antigen reactivity to the commercial pHSA.