Molecular Modification And Expression Of The Gene Of Kunitz Toxins From The Spider Ornithoctonus Huwena

BackgroundOrnithoctonus huwena is a kind of large poisonous spider endemic to China. Huwentoxin XI(HWTX-XI) is a Kunitz peptide toxin isolated from the spider. It has very strong trypsin inhibitory activity and can be used for the treatment of acute pancreatitis, and also plays a role in blocking the potassium channels, which shows weak neurotoxicity in animals. The two functional areas above are independent of each other, which facilitates toxin transformation. The new gene HW11c40is cloned from venom glands of the spider O. huwena, encoding Kuintiz peptide toxin. It has similar structure and the same critical acitve residue K14inhibiting the trypsin, but it does not have the key active residue R25inhibiting K+channels. HW11c40should have strong trypsin inhibitory activity with HWTX-XI, and its K+channel inhibitory activity would be even lower based on the bioinformational analysis. It is viable to eliminate HW11c40potassium channel blocking activity by genetically modification, accordingly to develop efficient drugs for the treatment of acute pancreatitis. However, the spider toxin separated from the natural venom of spider venom is far from being able to meet the needs of the research and development. Acquisition of spider toxin has been a perennial restriction for spider toxin research. At present, the expression of spider toxin by genetic engineering is an effective method of artificial obtain toxins.Objective1、Exploring the expression system suitable for the expression of HWTX-XI, to solve the problem of the research sources.2、Eliminating the neurotoxic side effects and improving the stability of the peptide by molecular transformation of HW11c40, to obtain specific and efficient trypsin inhibitor. To find a suitable expression system for HW11c40mutants, and will lay the foundation for screening suitable mutant to develop the novel highly efficient drug for acute pancreatitis and other trypsin-related diseases.Methods1、HWTX-XI fragment obtained by PCR was inserted into pET-40b (+) to construct recombinant vector, the recombinant vector was transferred into E. coli BL21(DE3) to obtain HWTX-XI fusion protein by periplasmic expression. Then, HWTX-XI fusion protein was digested by an enterokinase to release HWTX-XI, and was further separated and purified by using the combination Ni-NTA column and chromatography. Finally HWTX-XI was identified by the mass spectrometry. And the expression conditions were optimized.2、The Molecular of HW11c40was modificated by PCR site-directed mutagenesis, and then cloned in the vector pVT102U/a and pET-40b(+)to bulid [Y35C]、[Y35CD39N]、[RLY56(35) LEC] and [RLY56(10) (35) LETC] recombinant vectors repectively. The recombinant vectors transformed in Saccharomyces cerevisiae strain named S78and E.coli BL21(DE3) repectively to express protein. The express protein was identified by the mass spectrometry fainally after the further separation and purification.Results1、HWTX-XI fusion protein was expressed by prokaryotic periplasmic expression system, the target protein was successfully released by enterokinase digestion, and then verified by mass spectrometry. The maximum yield of target protein reachs3.4mg/L after lyophilized. The optimal expression conditions of HWTX-XI were cultured15-20h at30℃without IPTG.2、HW11c40was mutated successfully, and built the corresponding mutant recombinant vector. The Saccharomyces cerevisiae S78eukaryotic expression system, only expressed [Y35CD39N](HW11c40M2). All of the HW11c40mutants fusion protein were expressed by the BL21(DE3) prokaryotic periplasmic expression system, and then digested by enterokinase. Trince SDS PAGE confirmed that all the corresponding target protein were released by enterokinase digestion. The target protein of [Y35C](KpnIHW11c40M1) was further separated and purificated, and then further confirmed by mass spectrometry. Conclusions1、HWTX-XI has efficiently expressed in the pepriplasmic expression for the first time, and optimized in the expression conditions. A novel method of genetic engineering to express the toxin was established.2、The gene of HW11c40were modificated. Four mutants of HW11c40which may have more stable structure, lower potassium ion channel blocking activity and higher trypsin inhibitor activity were expressed in the pepriplasmic expression successfully. It is confirmed that the prokaryotic pepriplasmic expression was a suitable expression system for the expression of HW11c40mutants. Above all, all of these results lay a foundation for sreening valuable mutants, and then developing highly efficient drug for acute pancreatitis.