Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris.
The study describes expression and purification of recombinant hepatitis B small surface antigen (rHBsAg hereafter) in methylotrophic yeast Pichia pastoris strain GS115. For expression of the rHBsAg, a single copy of 678 bp cDNA was inserted at the unique EcoRI site downstream of the alcohol oxidase (AOX 1) promoter of the 8.2 kb pHIL-D2 vector. The cDNA-pHIL-D2 construct was used to transform the strain GS115, resulting in a Mut(S) (Methanol Utilizing Slow) phenotype in which the 226 amino acids containing active and full-length rHBsAg protein could be expressed intra-cellularly during slow growth and induction with methanol. The recombinant protein from the Mut(S) expressor was harvested by cell disruption, and purified first by adsorption-desorption on aerosil followed by two-step chromatographic separation i.e. anion exchange on DEAE resin followed by gel permeation on Superdex 75. Reversed passive hem-agglutination assay (RPHA) was used to test the antigenicity while SDS-PAGE was performed to check the purity of the 27 kDa rHBsAg and its aggregates. The results showed that disruption at 12 Kpsi (three cycles), or 30 Kpsi (1 cycle), desorption with 10mM carbonate buffer (pH 9-10), and storage at 4 degrees C without detergent did not adversely affect the antigenicity of the rHbsAg. However, the presence of detergents such as TritonX100 and deoxycholate in the disruption and desorption buffers, respectively resulted in reduced antigenicity during storage both at 4 and -20 degrees C in spite of higher initial yields.
Bardiya
Nirmala
N
Center for Advanced Bioseparation Technology Laboratory, Inha University, 253 Yong Hyun Dong, Nam Ku, Inchon-402751, Republic of South Korea. bardiyan@missouri.edu
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001709:Biotechnology / Q000379:methods; D004912:Erythrocytes; D006384:Hemagglutination; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000432:Methanol