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Prokaryotic Expression, Purification and Bioactivity Determination of Recombinant Human SCF
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Abstract:
Objective: To set up an optimal method of fermentation and purification of recombinant human stem cell factor (rhSCF). Methods: The effect of culture medium on the expression of rhSCF and the contents of rhSCF after different induction time were observed. The optimal condition for purification of rhSCF was also studied by changing pH, concentration of protein and chromatography procedure. The bioactivity of rhSCF was determined by UT-7 proliferation.Results: The expression level of rhSCF increased significantly in M9 culture medium containing glycerol, casein hydrolysate, yeast extract, peptone, and trace element. The optimal induction time for rhSCF expression was 6 h, approximately 30% of total protein. The insoluble inclusion body of rhSCF was denatured by 8M urea. After refolding for 24 h, the protein was firstly purified by acid precipitation and the supernatant was applied to an Source 30S column. The disulfide-linked dimeric rhSCF was excluded by reverse phase chromatography. The buffer was converted to PBS by S200. The purity of final product was over 95% with the biological activity more than 6×10 5 U/mg. Conclusion: The optimal condition for rhSCF preparation was successfully established, which can be used for scale-up production in the future.
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