Studies On The Transduction Peptide: Expression In E.coli And Its Activity In Vitro

Several proteins can traverse biological membranes through protein transduction. Small fragments of these proteins and their derived polypeptides, which contain a relatively high number of positively charged amino acids, can transduce almost all molecules, such as proteins, peptides, antisense nucleic acids, or as in-frame fusions with full-length proteins into cell membrane and locate at certain organelles in a receptor- and transporter- independent fashion. It is important to develop the new delivery systems for the study of enzymes, cytokines, especially those playing an important role in intranuclear processing, and for the intracellular addressing of pharmacological substances.Naturally occurring polyclonal anti-DNA antibodies from spontaneous lupus erythematosus(SLE) penetrated and accumulated in the nuclei of a variety of cultured cells. Based on the sequence of combined CDR2 and CDR3 of the heavy chain variable regions of the mAb, named TP, the whole nucleotides were synthesized. After sequencing, the TP was cloned into an expression vector pMTY4 in a fusion fashion. The fusion expression vector of TP and TFAR19, a TF-1 apoptosis related novel gene, was also constructed. MS₂-TP and MS₂-TP-TFAR19 fusion proteins were effectively expressed after induced in E.coli. pop2136. The inclusion bodies of the two fusion proteins were denatured, refolded and purified before activity assay.The purified MS₂-TP fusion protein was added to cultured 3T3 cell monolayers. One hour later, intracellular MS₂ transduced using TP can be detected with Western blot,while no positive band in control group MS₂. The purified MS₂-TP and MS₂-TP-TFAR19 fusion proteins were added tocultured HL-60 cells at different gradients. The transduction ability of TP was detected with FACS. Compared with MS₂ and MS₂-TP, it was found that TP transduced exogenous TFAR19 into cells and obvious cell apoptosis can be detected.The mixture of purified MS₂-TP and pEGFP were added to the culture of 293T cells. MS₂-TP-pEGFP penetrated into cells and green fluorescence was found with fluorescence microscope, whereas no green fluorescence was observed in the control group only pEGFP added.The cell penetration assays indicated that TP was sufficient for efficient cell transduction and nucleus translocation of in-frame fusion protein in a rapid, time- and concentration- dependent manner. TP can bind and deliver plasmids into nucleus and expressed protein. TP may provide a versatile efficient import system for bio-molecules including functional proteins, DNAs and oligonucleotides. TP could be applied in the study of signal transduction as well as gene therapy.