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| Prokaryotic Expression and Purification of Human SCYL1-BP1 and Its Identification |
| CHEN Xiao-jing, CHEN Xiao-mei, WANG Yang, SHI Hui-li, HUO Ke-ke |
| State Key Laboratory of Genetic Engineering, College of Life Sciences, Fudan University, Shanghai 200433, China |
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Abstract Previous studies revealed that human SCYL1-BP1 gene plays an important role in the cell cycle and tumor suppression. Objective: Construction of recombinant human SCYL1-BP1 gene expression system in prokaryotic strain, in order to obtain sufficient purified SCYL1-BP1 protein for the pharmacology experiments.Methods: The full length coding sequence of SCYL1-BP1 was cloned from human placenta cDNA library by PCR and inserted into plasmid pET-28b-SUMO, resulted in the recombinant prokaryotic expression plasmid pET-28b-SUMO-SCYL1BP1. Then it was transformed into E. coli strains BL21(DE3) and Rosetta(DE3) for expression using IPTG as an inducer. The expressed recombinant protein was purified by Ni-NTA chromatography and identified by SDS-PAGE and Western blot with anti-His-tag and anti-SCYL1BP1 monoclonal antibody.Results: The recombinant prokaryotic expression strain BL21(DE3)-pET-28b-SUMO-SCYL1BP1 was constructed successfully. SDS-PAGE and Western blot results showed that the fusion protein was 65 kDa in soluble form, which can be recognized by anti-His-tag antibody and anti-SCYL1-BP1 monoclonal antibody.Conclusion: The recombinant human SCYL1-BP1 gene was successfully expressed in the E. coli strain BL21(DE3), and the purified SCYL1-BP1 protein was identified correctly by anti-SCYL1BP1 monoclonal antibody. The results provided a favorable condition for further study on SCYL1BP1 function and application.
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Received: 08 May 2012
Published: 25 September 2012
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