title: Preparation of cell-permeable Cre recombinase by expressed protein ligation.
abstract: BACKGROUND
  Protein 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.
  
  
  RESULTS
  Expressed 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.
  
  
  CONCLUSIONS
  Collectively, 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.
authors.affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin, 449-791, Republic of Korea. helhela@naver.com.
authors.forename: Soo Kyung
authors.initials: SK
authors.lastname: Lyu
authors.affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin, 449-791, Republic of Korea. hmkwon@hufs.ac.kr.
authors.forename: Hyockman
authors.initials: H
authors.lastname: Kwon
mesh_terms: D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047668:Inteins; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D057846:Cell-Penetrating Peptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C015462:polyarginine; D001710:Biotin; C045073:Cre recombinase; D019426:Integrases