Cloning And Expression Of The Extracellular Domain Of Fas Ligand, Screening And Characterization Of Antisense Peptides Of Fas

[Background] The conserved, universal code defines the primary amino acid sequences of all proteins of all organisms. However, this code has been thought to be limited, unable to provide additional information appropriate to defining the three-dimensional structure and function of these proteins. Research to date suggests that sense and antisense peptides coded for by mutually complementary nucleic acid sequences are capable of interacting specifically, thereby suggesting the existence of a second, two-dimensional genetic code (i.e.,proteomic code ) . Mekler Idlis ( M-I) pair theory suggests that each codon-directed amino acid residue in a sense peptide may make a specific pair-wise interaction with the corresponding complementary codon-directed residue in the complementary peptide.AHBs theory suggests also the parts between/in the proteins that are capable of interacting specifically.The interaction between receptor and ligand is the recognition and interaction between proteins.If the receptor is the sense peptide,the ligand , which can specifically bind to it ,must have one or several antisense peptides.These antisense peptides muat be located at the key place which has relationship with the function of the ligand.Fas (also known as APO-1 or CD95) , the receptor for FasL, belongs to the TNFreceptor family. A cell bearing Fas can be set on a cell death (apoptosis) pathway when the ligand FasL binds to Fas. Using a computer program, a peptide was deduced from residues 256-265 of human Fas ligand, based on the hypothesis that it should contain a specific binding site to the corresponding Fas. This short peptide and Fas contain 70% of sense-antisense pairs. As a result, they should interact specifically. Thereof the ten-peptide is chemically synthesized, and its apoptosis-inducing activity on tumor cells bearing Fas is investigated in parallel with recombinant excellular domain of FasL (FasL-ECD) . Thepresent investigation is favorable for screening the new generation of anti-tumor peptides. [Objective] (1) To clone and express FasL-ECD in E.coli. The recombinant FasL-ECD was purified and its biological activity was analyzed on several cell lines and the most sensitive cell lines are selected. (2) Using a computer program, a single short peptide is derived from antisense homology box of Fas ligand and is chemically synthesized. (3) Examine the apoptosis-inducing effect of the recombinant FasL-ECD and the ten-peptide on the most sensitive cell lines, and the relationship between them was analysed. [Methods] By using the overall RNA of our previously cultured human melanoma cell line (A375) , full length FasL gene is detected by RT-PCR. Using the cDNA as template,.the extracellular domain of FasL (FasL-ECD, 127-278aa) is amplified by PCR. The PCR products are directly cloned into T vector pMD-18T. One recombinant clone,pMD-T-FasL-ECD,are obtained and sequenced. FasL-ECD coding sequence is subcloned into pET-lla expressing vector, recombinant expression vector are named as pET-FasL-ECD. This plasmid is introduced into E. coli BL21. After induction with 1mmol/L IPTG , the protein expression is analyzed and confirmed by coomassie-stained SDS-PAGE. The recombinant FasL-ECD protein is purified and its bioactivity is assayed on tumor cell lines including human 1990 (pancreatic carcinoma), HepG-2 (hepatocellular carcinoma), M85 (gastric cancer) , HL60 (human promyelocytic leukemia) , NCI-H460 (non-small cell lung) , PANC-1 (pancreatic carcinoma) , and K562 (human leukemia) . The most sensitive cell lines are selected out.These cell lines are cultured in RPMI 1640 medium containing 10% fetal bovine serum in a humidified atmosphere of 5% Co2 at 37 .A computer program was developed for searching of antisense peptide to Fas from Fas ligand, and a peptide deduced from residues 256 to 265 (N )-HLYVNVSELS-(C)of humanFas ligand was screened out. The peptide is synthesized in Shanghai Shenyou Biological Technology Ltd.and Sangon Biological Company.The most sensitive cell lines,NCI-H460 and HepG2 are...