The new method for transfection of macromolecules into cells using protein transductions domain

( views:322, downloads:0 )
ZHANG Yan-jun(Institute of Hematology,CAMS & PUMC, State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
REN Si-mei()
LU Hong()
LIU Qian()
ZENG Jie()
ZHANG Yi-zhi(Institute of Hematology,CAMS & PUMC, State Key Laboratory of Experimental Hematology, Tianjin 300020, China)
Journal Title:
Journal of Leukemia & Lymphoma
Volume 21, Issue 10, 2012
Key Word:
Protein transductions domain;Transfection

Abstract: Objective To construct a fusion protein to transfect some cell lines that were difficult to be transfected such as neoplastic cells, nerve cells, stem cells. Methods PCR was performed to amplified protein transductions domain(PTD),G4S and streptavidin (Strep).Enzymatic digestion and ligation were used to construct pAYZ-PTD-Strep plasmid. Fusion protein was induced to express by AP5 medium and was isolated by E-tag affinity chromatography. Fusion protein was identified by Western blot. eGFP was trasfected into U937 cells by pAYZ-PTD-Strep. FACS was performed to detect transfection percentage. Results Fusion protein PTD-G4S-Strep was expressed as soluble protein.The concentration of fusion protein was about 0.7 mg/L,and purity was over 90 %. The protein could carry plasmid into a suspended cell line, U937 cells. The transfection-efficiency of protein was higher than monometer PTD.Conclusion The protein PAYZ-PTD-Strep could carry macromolecules into blood tumor cells,and its biological activity may be expected to develop into a highly efficient and reliable transfection method.

  • [1]任思楣,张砚君,彭洪薇,等.靶向白血病干细胞CD123的毒性融合蛋白的制备.白血病·淋巴瘤,2011,20:490-493.
  • [2]曹阳,黄晓园,庄亮,等.组蛋白去乙酰化酶抑制剂提高腺病毒对K562细胞转染及杀伤效率的研究.白血病·淋巴瘤,2007,16:1-6.
  • [3]Becker-Hapak M,McAllister SS,Dowdy SF.TAT-mediated protein trailsduction into mammalian cells.Methods,2001,24:247-256.
  • [4]Ho A, Schwarze SR, Mermelstein SJ, et al. Synthetic protein transduction domains:enhanced transduction potential in vitro and in vivo.Cancer Res,2001,61:474-477.
  • [5]Lewin M, Carelesso N, Tung CH, et al. TAT peptide-derivatized magnetic nanoparticles allow in vivo tracking reeovery of progenitorcells.Nat Biotechnol,2000,18:410.
  • [6]Torchilin VP,Levchenko TS.TAT-liposomes:a novel intracellular drug carrier.Curr Protein Pept Sci,2003:4133-4140.
  • [7]Tseng YL, Liu JJ, Hong RL, et al. Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat: a kinetic and efficacy study.Mol Pharmacol,2002,62:864-872.
  • [8]叶南慧,林艳云,潘建茹,等.PTD介导的蛋白转导技术及其在医学领域中的应用.生物医学工程学杂志,2011,28:41-44.
  • [9]Umezawa N,Gelman MA,Haigis MC,et al.Translocation of a betapeptide across cell membranes.J Am Chem Soc,2002,124:368-369.
  • [10]Futaki S, SuzukiT, Ohashi W, et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.J Biol Chem, 2001,276:5836-5840.
WanfangData CO.,Ltd All Rights Reserved
About WanfangData | Contact US
Healthcare Department, Fuxing Road NO.15, Haidian District Beijing, 100038 P.R.China
Tel:+86-010-58882616 Fax:+86-010-58882615