The Expression And Transduction Efficiency Of Cell-permeant Cre Fusion Protein

Through the medium of protein transduction domain(PTD) composed by several alkalinepeptides, the functional proteins linked covalently with DNA, peptides, proteins or othermacromolecules can penetrate the membranes of mammalian cells, even the blood-brainbarrier and transfer unconventionally through unclassical pathway. The cationic11-aminoacid fragment derived from human immunodeficiency type1(HIV-1) TAT protein, termedTAT protein transduction domain, has proven to be an invaluable tool to deliver a wide varietyof macromolecules penetrate the plasma membrane and into intact tissues.The Cre/loxP site-specific recombination system, derived from bacteriophage P1, hasbeen widely used for the DNA recombination research on prokaryote and eukaryote both invitro and in vivo. The high specificity and efficiency of the system make it play an importantrole in many regions, such as cloning tools, gene trapping, removal of specific genes,chromosome engineering and targeted integration.In order to investigate transduction efficiency of different kinds of TAT and/or NLSmodified Cre fusion proteins,11prokaryotic expression vectors were constructed: The codingsequence(CDS) of Cre gene was cloned into pET-28a(+),then TAT and NLS sequences weresynthesized and inserted at the N terminal or/and C terminal of CDS of Cre gene inpET-28a(+).After confirmed by restriction enzyme digestion and sequencing,11expressionvectors were used to transform E.coli BL21(DE3),respectively. After induced by IPTG,11His-tagged Cre fusion proteins were expressed and purified by Nickel-affinity column.293-C31-L2GFP cell line was used to detect the transmembrane function and biology activityof the Cre fusion proteins, and the transduction efficiency was measured by FACS. The resultsshow：1.The open reading frame sequence of Cre gene were cloned successfully by PCRmethod from plasmid pCAG-Cre-IP. The open reading frame sequence of target gene werecloned into pMD19-T vector by using gene recombination technology. E.coli (DH5α) weretransformed with the recombinant plasmids.Target gene is connected in the prokaryoticexpression vector pET-28a (+) cloning site after sequencing right. Build11prokaryoticexpression vector containing the Cre TAT and NLS sequence2. Using0.7mM IPTG induced7h at28℃,11prokaryotic expression vector in E. coliBL21(DE3) prokaryotic induced expression. Histidine affinity chromatography purifiedfusion protein and by Western blot, and finally got the high purity Cre fusion protein. 3.Cell-permeant results show that among11different kinds of TAT and/or NLS modifiedCre fusion proteins, the HNTC (His-NLS-TAT-Cre) protein, with an excellent combination oftransmembrane function and biology activity, can serve as efficient tools that are available tomanipulate gene in mammalian genome.