Preparation of cell-permeable Cre recombinase by expressed protein ligation

BMC Biotechnology

Research Article

Hyockman Kwon¹  Soo Kyung Lyu¹ 

[1] Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, 449-791, Yongin, Republic of Korea;

关键词: Expressed protein ligation;    Cre recombinase;    Polyarginine;    Cell-penetrating peptides;    Intracellular delivery;   

DOI  :  10.1186/s12896-015-0126-z

 received in 2014-11-10, accepted in 2015-02-09,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundProtein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.ResultsExpressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.ConclusionsCollectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

【 授权许可】

CC BY   
© Lyu and Kwon; licensee BioMed Central. 2015