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Gloning、Expressing、Purification Of PyIC And Chemical Synthesis Of Cyclic Peptide SFTI-1

Posted on:2015-02-14Degree:MasterType:Thesis
Country:ChinaCandidate:Y Q ChenFull Text:PDF
GTID:2250330428465539Subject:Biochemistry and molecular
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Paper will begin with introduction of two parts. The first part is pyrrolysine protein molecular cloning and recombinant expression, the second part is chemical synthesis of the cyclic peptide drugs SFTI-1.Analysis and identification the protein structure and function of PylC is important to clarify its mechanism to catalytic pyrrolysine synthesis, revealing the specific catalytic reaction mechanisms and substrate ATP interaction, to provide the support for clarify pyrrolysine concrete synthesis reaction steps, which will give help to understand the exact mechanism pyrrolysine synthesis.Py1(pyrrolysine) is the22amino acids encoded by the native gene discovered in recent years, In the case of methane-producing bacterium the specific methyl transferase enzyme (MtmB, MtbB, MttB) and its specific substrate MMA, DMA and TMA reaction of methane and carbon assimilation. This highly expressed nonhomologous gene encoding the methyltransferase in the synthesis of protein of whole sequence, Succinyl UAG codon encoding frame not to formation a transcriptional termination but encoded a pyrrolysine. These phenomenon was first found in methane methyltransferase (MtmB), later the same phenomenon were found in methane methyl transferase (MtbB) methane and three methyl transferase (MttB), although these gene sequence have none homologous, but all of them are encoded UAG to generates pyrrolysine. Ancient methane-producing bacterium pylTSBCD gene has been transferred in E. coli, in the progress of protein synthesis pylTSBCD endogenous gene can be incorporated to synthesis pyrrolysine in protein. UAG encoded into pyrrolysine need pylT4-6produces tRNAPyt (also known as tRNAcuA) andpylS encoding synthetic pyrrolysine tRNA synthetase Py1S. py1B, py1C and pylD are three independent genes which are essential in tRNA genes of pyrrolysine synthesis. In the translation process, pyrrolysine synthesis like the other20kinds of amino acids into the same sequence in such a manner, Pyl connected to the homologous py1T derived by specific amino tRNA-tRNA synthetase pylS. With reasonably speculate here on the synthesis of pyrrole lysine:From Py1B, Py1C and Py1D three consecutive enzymatic reactions of the two lysine synthesis pyrrolysine. The recently reported Py1B is a TIM barrel protein, Lysine conversion to the3R-methyl-D-ornithine (3MO) via a free radical mechanism, the intermediate molecules3MO connected to of lysine s-amino group, by an ATP-dependent formation of isopeptide bonds with Py1C. Finally, the product was catalyzed oxidation of L-lysine hydrochloride Nε-3R-methyl-D-ornithine by Py1D.Cyclic peptides, which are generally referred to peptides with an end-to-end or head-to-tail linkage of the peptide backbone through an amide bond, have been considered as useful tools in pharmaceutical science and chemical biology. Compared to their linear counterparts, the cyclization of peptide has generated conformational constrained analogues with improved bioactivity, selectivity, bioavailability and higher receptor-binding affinities. Sunflower trypsin inhibitor (SFTI-1) is a naturally occurring cyclic pe ptide with only14residues, which is regarded as one of the smallest and most potent trypsin inhibitor as known. Since its discovery, SFTI-1has attracted much interest because of its small size and high potency, which make it an ideal candidate as a platform for the design of novel small proteinase inhibitors. However, the biosynthesis and peptide cyclization mechanisms for SFTI-1still beyond the complete understanding, which has restricted their production by genetic approaches. So we developed of chemical synthesis, chemical synthesis method including Boc and Fmoc method, Fmoc method is more common. Based on this, many groups have reported the synthesis of SFTI-1using solid-phase Fmoc chemistry with the latter cyclization of SFTI-1in solution or an on-resin cyclization approach. In spite of these improvements, efforts are needed to overcome current constraints, e.g. relative long time (16-24h), high temperature and time-consuming steps for the synthesis of thioester and its surrogate for cyclization. Herein, we report a high efficient and time-efficient method of the synthesis of SFTI-1via the ligation of peptide hydrazide. Take the advantage of this hydrazides based native chemical ligation, we found that SFTI-1can be synthesized with increased.
Keywords/Search Tags:pyrrolysine, PylC, cyclization peptides, SFTI-1, peptide hydrazide
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