Study On CPPs- Mediated Delivery Of Target Protein Into Biomembrane

Biomacromolecules play an important role in the treatment of many diseases, such as SOD, CAT, VEGF, wonder drug for diabetes, insulin, anti-oncogene expression inhibiting the growth of cells or anti-tumor proteins (p53, p27) contributing to the apoptosis and death of tumor cells. As a result of cell membrane serving as the natural barrier, only the small molecular compounds or organisms whose molecular volume is smaller than 600Da, non-polar hydrophobic, fat-soluble or having a carrier on the cell membrane can get through cell membrane and enter the cell. For years, researchers are trying hard to explore effective ways to transduce the efficacy molecules into the viable cells. Although the methods such as electric perforation, microinjection, changing the physical and chemical state of large molecules, gene transfection granular (as liposome) pack have many advantage, they are complicated to manipulate, with low efficiency and difficulty in adjustment. Many healing medicine yet no function of biomembrane penetration , easy degradation and hydrophilic molecules greatly restricted in their application in such research fields as cytobiology and pharmacology.In recent years, series of sequences of amino acid are found to have the cell-penetrating function, with the length several amino acids or tens of amino acids, most of length being less than 20 amino acids, called cell-penetrating peptides, CCPs. CCPs, also called protein transduction domain, PTD or Trojan horse peptides, is able to through the use of non-receptor-intervention and non-energy consuming methods, effectively introduce segments of protein, polypeptides, nucleic acid into the cells of many mammal animals and it will not within the specific concentration lead to cell damage. The discovery of CPPs sheds bright light on the use of biomacromolecules in treating diseases. It has a very good applicable prospect in such research fields as cytobiology, gene-therapy, drug transduction in vivo, evaluation of clinical medicine and medical immunology. There are two kinds of CPPs: natural and synthetic. The study of CPPs began in 1988 when Green and Frankel proved that the trans-acting protein factor of HIV- I can activate Tat to penetrate into cytoplasm and nucleus. In 1977, Vives et al found in the HIV-TAT, there was a close correlation between an abundant alkaline amino acids, segments of polypeptides carrying positive charge and protein transducing function. Natural CPPs are now already found to be of three types, coming from HIV-1 & SIV of TAT, drosophila allotypic transcription factor, ANTP and transcription factor of HSV-I, VP22. The CPPs are polypeptide segments of various lengths carrying positive charge, rich in such alkaline amino acid residues as arginine, lysine and so on. By making good use of this feature, synthesized polyarginine and lysine also have cell-penetrating activity. The strains constructed by 9 arginine residues and 9 lysine residues have more powerful protein transducing activity than TAT PTD. Through the study of natural CPPS, Morris group designed and synthesized a 21- amino-acid-residue amphoteric peptide, PEP-1 (KETWWETWWTEWSQPKKKRKV). PEP-1 is different from natural CPPs. It doesn' t necessarily have to combine aim biomacromolecules with covalent bond to make the most of exogenous biomacromolecules, carrying exogenous protein effectively into cells, and thus greatly changing the past situation of CPPs' s defect of carrying only effective transport denatured proteins.Green fluorescence protein, GFP consists of 238 amino acids, whose molecular volume is 27kD and under the activation of 395nm light wave, GFP can give out bright green fluorescent light. Its expression doesn' t have any specificity; It doesn' t need any helping factor, substrate or coordination of other gene-expression; it has high sensitivity. In most of cells, GFP fluorescence is evenly distributed everywhere in the cytoplasm, nucleus and far end process such as axon, which makes it possible to conduct research directly on the viable cells with the help of fluorescent microscope. As a reporting protein, GFP is widely applied in detecting aim gene expression, studying endocelluar substance metabolism and tracking the differentiation of cell system. Enhanced green fluorescence protein, EGFP is the mutant of GFP, deriving from the 64^th and 65^th phenylanlanine and serine exchanged into leucine and threonine respectively. Under the activation of 480nm wave length, EGFP can give out 35 times brighter green fluorescence, making it easier and convenient to observe and detect.P27 is an anti-oncogene, discovered by Polyak and others in 1994. Its roles in the development of ontogenesis and its value in the treatment of tumors have been noticed. Under normal conditions, when cells enter stage S and p27 gene expression drops, the levels of G0/G1 are raised. P27 protein is cyclin dependent kinase (CDK) inhibitors (CDKIs) acting as a negative control factor of cell cycle, which plays a role during the whole cycle. It can suppress the activity of many kinds of CDK-cyclins and CDKIs by binding them to delay the cells in GO or G1, or prevent the progressing from G1 to S, thus to prevent the proliferation of cells. P27 has been widely used in the research of the therapy of many tumors such as alimentary tumor, mammary tumor, lung cancer and cervix tumor. Moreover, it can regulate the drug resistance of the tumor, protecting against organism' s injury from inflammations. P27' s degradation mainly results from the ubiquitin - Proteasome intervention of threonine' s phosphorylation at position 187. Its half declining stage is short (about 2hours), and is unstable in vivo, as a result, P27kipl is also referred to as "short-life protein" . Kudo' s group and others found out that P27 will not be activated by ubiquitin - Proteasome and protein' s degradation will fall significantly if threonine will be replaced by alanine. Jean and others transfected p27 into HeyC2 cells and discovered the growth of the cells were significantly inhibited.P27mt used in the present study is natural p27 or called wild p27wt, a protein encoding thronine at postion 187 and cDNA at position 188, with the sequence of ACGCCC mutated into ATGATC. After mutation, p27mt loses the hydroxyl and cannot be phosphorylated, its half declining life is prolonged, resulting in higher binding ability with CDK-cyclins and higher anti-tumor effect. As 27KD protein consisting of 198 amino acids, P27mt can hardly penetrate through lipid bilayer and reach therapeutic concentration in cells by itself. The present research is composed of three sections. The first Section: Study on the penetrability of TAT-EGFP fusion protein for biomembranes of miceSynthesized double-stranded oligomeric nucleotide encoding 11-amino acid HIV-TAT PTD, amplified aim gene EGFP by polymerase-chain reaction and used molecular cloning routine operations to construct pET28a-TAT-EGEP recombinant express -ing carrier, transformed host bacteria E. coli BL21(DE3) and induced by IPTG with TAT-EGFP for fusion protein expression, the purified fusion protein was obtained by Ni²⁺ affinity gel column chromatography and the penetrability of TAT-EGFP fusion protein under a fluorescence microscope after intraperitoneal injection of the recombinant protein was observed. The research shows that TAT-EGFP quickly reached all tissues in 30min and reached maximum intracelular concentrations in 4 hours after intraperitoneal injection. The research proves that fusion protein resulting from TAT-PTD and EGFP combination can penetrate into all biomembranes. The finding provides a good reporting protein in the studies of TAT mediated- other macromolecules to penetrate biomembranes and at meantime a bright prospect for the application of biomacromolecules in the clinical therapy. The second section: Study on the penetrability of PEP-1-EGFPSynthesized double-stranded oligomeric nucleotide encoding 21-amino acid PEP-1, amplified aim gene EGFP by polymerase-chain reaction, and used T/A cloning routine to construct pGEM-T-EGEP recombinant plasmid, and used molecular cloning routine operations to construct pET15b -pep-1-EGEP recombinant expressing carrier, transformed host bacteria E. coliBL21(DE3) and induced by IPTG with PEP-1-EGFP for fusion protein expression, the purified fusion protein was obtained by Ni²⁺ affinity gel column chromatography and the study of the penetrability of PEP-1-EGFP fusion protein on mice in vivo and through culturing cells in vitro was conducted. The experiment shows that the application of pGEM-T easy carrier makes the process of gene cloning easier and more convenient, and more accurate in construction. By adding PEP-1 to the N end of EGEP to synthesize the fusion protein of CPPs, it has the advantage of no complicated process of mutation and renaturation and less toxic action. It can penetrate through cells cultured in vitro and all the tissue organs in mice in vivo in the form of natural activity. It has laid a solid experimental foundation for the safer and more effective use of penetrating peptides.The third section: Study on the penetrability of PEP-1-P27mt Synthesized double-stranded oligomeric nucleotide encoding PEP-1 peptides, constructed prokaryotic expression pET15b-pep-1-p27mt recombinant, transformed E.coli BL21 (DE3)pLysS and induced with IPTG for fusion protein PEP-1-P27mt expression. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 10min and reached maximum intracellular concentrations in 2h. PEP-1-P27mt of 1mmol/L terminal concentration has most strong activity in the inducing apoptosis. After injecting PEP-1-P27mt into the SD rats' abdominal cavity, immunofluorescence test showed that fluorescence was found in all the major organs 30min after the injection and the value of fluorescence in all the tissues reached the peak in 4h. This research demonstrates that PEP-1 can effectively carry P27mt into the cultured cells and into animal bodies. It provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in inducing apoptosis of tumor cells.