Recovery of choline oxidase activity by in vitro recombination of individual segments |
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Authors: | Birgit Heinze Nina Hoven Timothy O’Connell Karl-Heinz Maurer Sebastian Bartsch Uwe T Bornscheuer |
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Institution: | (1) Laboratory of Molecular Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, Jiangsu, People’s Republic of China;(2) Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, Jiangsu, People’s Republic of China;(3) The First Clinical College, Nanjing Medical University, Nanjing, 210097, Jiangsu, People’s Republic of China, 210029 |
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Abstract: | Elevated levels of B cell-activating factor of the TNF family (BlyS) have been implicated in the pathogenesis of autoimmune
diseases in human. Removal of pathogenic B lymphocytes by decoy receptors has demonstrated clinical benefit in both oncological
and immunological diseases. In this report, we have constructed vectors for the convenient and rapid expression of the extracellular
domain of BR3(sBR3) fused to the Fc fragment (hinge, CH2, CH3) of human IgG1 in the methylotrophic yeast, Pichia pastoris. SDS-PAGE assays of culture broth from a methanol-induced expression strain demonstrated that the recombinant sBR3-Fc fusion
protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The recombinant protein
was purified to >95% using protein A affinity chromatography and size exclusion chromatography steps. Bioactivity of the recombinant
sBR3-Fc was confirmed by the ability of the protein to inhibit mouse B lymphocyte proliferation induced by BLyS in vitro.
Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional sBR3-Fc fusion protein for both research and
industrial purposes. |
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Keywords: | BR3 IgG1 Fc Fusion protein Pichia pastoris Secretion expression Protein purification |
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