Study Of Methods Of The Seamless Gene Cloning And The Expression And Purication Of Active Peptides

Ion channels?receptors?,such as voltage-gated,mechanical gated and ligand-gated channels,play important roles in many physiological activities.Abnormal function or dysfunction of ion channels?receptors?are involved in many diseases such as chronic pain,heart or brain diseases,arthritis and dyskinesia.Natural polypeptides?2-50 amino acids?can serve as regulatory molecules that interact with ion channels?receptors?.The two most important rate-limiting steps in the study of high-throughput activity of active peptides are:1)whether or not large numbers of expression vectors can be constructed in less time by using this gene cloning method;2)whether or not recombinant polypeptide with correct disulfide bonds can be obtained by using this expression system.In this thesis,we first established a new gene cloning method.This strategy is named OEPR cloning,which is based on Overlap Extension PCR and Recombination in vivo.The most important feature of OEPR cloning is the perfect combination of overlap extension PCR strategies and homologous recombination-based methods.OEPR cloning was performed by introducing a pair of primers to amplify the insert genes.the 5'end of each of the upstream primer and the downstream primer own an overhang that is homologous to the sequences of insert sites of the vector,and have15-30 bases and Tm values of approximately 68-70?,respectively.After two steps of PCR reaction,a linear plasmid,which has a homologous sequence?15-30 bp?at both ends,can be obtained and repaired into a completely circular plasmid in E.coli in vivo.A large fragment?1-6 kbp?can be efficiently inserted into the vector?pGADT₇,7988 bp?by using OEPR cloning.When the homologous recombination arm is 30 bp,the positive rate can reach about 80%.Multiple large fragments?two 1 kbp,two 2 kbp,two 3 kbp,three 1 kbp,three 2 kbp and four 1 kbp genes?can also be efficiently and simultaneously assembled into the vector?pGADT₇,7988 bp?by using OEPR cloning.When the homologous recombination arm is 30 bp,the positive rate can reach about80%.OEPR cloning is an efficient and time-saving seamless cloning approach that successfully integrates single large fragments or multiple fragments into the vector.Some of the plasmid-based gene manipulations such as gene mutation,gene chimera construction,multi-gene fusion can be performed efficiently using OEPR cloning.In addition,this thesis also established a new secretion expression and purification system in E.coli,which can be used to express and purify cysteine-rich active polypeptide.The open reading frame of the system includes signal peptide,6×His purification tag,SUMO fusion tag and toxin molecule.The signal peptide is the mutant M5 of signal peptide derived from the cyclomaltodextrin glucanotransferase in Bacillus subtilis G1 strain.The SUMO fusion tag was used as both a solubilizing tag and a restriction site in this study.The ULP1 kinase can efficiently recognize the structure of the SUMO protein and cleave at the C-terminus end of the two glycines.So the digestion doesnot introduce extra amino acids into the toxin molecules.To test the effect of this secreted expression system,we selected the toxin molecules HWTX-I,which is from Ornithoctonus huwena and has 33 amino acid residues and three pairs of disulfide bonds,and HNTX-IV,which is from Selenocosmia hainanum and has 35 amino acid residues and three pairs of disulfide bonds.Firstly,we inserted the G1M5 signal peptide into the 5'end of the 6×His tag in pET-SUMO vector to construct the intermediate vector pSE-G1M5-SUMO by OEPR cloning method,and then we used OEPR cloning method to insert the toxins HWTX-I and HNTX-IV into the 3'end of the SUMO fusion tag in the pSE-G1M5-SUMO vector to construct the secretory expression vectors pSE-G1M5-SUMO-HWTX-I and pSE-G1M5-SUMO-HNTX-IV.Then,the secretory expression plasmids were transformed into E.coli BL21?DE3?,respectively,and the fusion proteins were expressed by auto-induction expression system and then secreted into the culture medium.The fusion recombinant protein was enriched by weak cation exchange resin from the supernatant of the autoinduced culture by centrifugation,followed by affinity purification through Ni-NTA resin,and then was concentrated and desalted by ultrafiltration.The toxins HWTX-I and HNTX-IV were released after the digestion by the protease ULP1,and re-purified by RP-HPLC to obtain the corresponding peak under the absorption of 215 nm.The molecular weights of rHWTX-I and rHNTX-IV,which were identified by MALDI-TOF mass spectrometry,were 3750.8 Da and 3987.6 Da,respectively,which were consistent with their theoretical molecular weights of 3750.43 Da and 3986.58 Da,respectively.The electrophysiological activity of the rHWTX-I and rHNTX-IV were detected by the whole cell patch clamp system.10?M of rHWTX-I and rHNTX-IV inhibited more than90%of currents of hNa_v1.7 expressed on HEK293 cells,and the IC₅₀ values of rHWTX-I and rHNTX-IV were 500 nM and 126 nM,respectively,which were consistent with its natural physiological activity?630 nM and 34 nM,respectively?.The above experimental results show that this secretion expression system toxin molecules HWTX-I and HNTX-IV with the correct disulfide bonds can be successfully expressed and purified.This secretory expression and purification system can be applied to the expression and purification of other cysteine-rich polypeptide toxins.