Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli.
Overexpression of rhIFN-alpha2b was obtained by synthesizing a codon optimized gene for IFN-alpha2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNalpha, which had the IFN-alpha2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible lambdaP(L) promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2g/L at a final OD(600) of 67. At this point, the IBs represented approximately 40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of approximately 3g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of approximately 3 x 10(9)IU/mg protein.
Srivastava
Poonam
P
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India.
Bhattacharaya
Palash
P
Pandey
Gaurav
G
Mukherjee
K J
KJ
D001681:Biological Assay; D019149:Bioreactors; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007218:Industrial Microbiology; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D003470:Culture Media; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins