Cloning, Expression And Purification Of Fusion Protein Genes As Well As Their Function Research

In this work, we decided to develop some carriers of therapeutic molecules to the nuclei of cells. Two carriers were studied here. One is TAT-NLS which is derived from human immunodeficiency virus I ( HIV-I ) and Simian Virus SV40 large tumor antigen (SV40 T-ag) respectively. TAT-NLS was cloned into pET-22b(+) vector via Nde I and Not I restriction sites, resulting in pET-TAT-NLS. The report gene, enhanced green fluorescent protein (eGFP) was generated by PCR from pEGFP-C1 vector, using the following primers synthesized: 5'-GCGGCCCAGCCGGCCATGGTGAGCAAGGGCGAG-3', 5'-GCGCGGCCGCCTTGTACAGCTCGTCCATGCCGA-3'.eGFP gene was then added to the end of TAT-NLS via Sfi I and NotI. After the successful construction of pET-TAT-NLS-eGFP vector encoding TAT-NLS-eGFP , it was used to transform Escherichia colistrain BL21(DE3), allowing isopropyl β-D-thiogalactoside (IPTG)-inducible expression of His-tagged proteins. The other one is F3 peptide sequence that encodes an N-terminal fragment of human high mobility group protein 2 (HMGN2, formerly HMG-17). Using similar ways we constructed a recombinant vector pET-F3-eGFP, which was then transformed into Escherichia coli strain BL21(DE3) and induced to express recombinant protein by IPTG. After successful purification of the two recombinant proteins TAT-NLS-eGFP and F3-eGFP, the expressed proteins were used in cell transduction experiment in vitro.It was found that these proteins accumulated in the nuclei of experimented tumor cells after incubated with tumor cells. Thus, TAT-NLS and F3 can carry a payload (phage, fluorescein[1], protein) into tumor cell cytoplasm and nuclei. These peptides maybe suitable for targeting proteins, cytotoxic drugs and gene therapy vectors into tumor cells.