The Optimization Of Fermentation Conditions And Application Of The Antifreeze Protein From Microdera Puncitpennis

Antifreeze protein (antifreeze protein, AFP), also known as thermal hysteresis protein, has three basic characteristics:thermal hysteresis effect (THA), the effect of ice crystals form, recrystallization inhibitory effect (RI), To date, AFPs have been widely found in a variety of organisms,such as fish, insects, plants, bacteria and fungi,isolated from a number of fish,plants, bacteria, fungi and arthropods.But studies show insect AFPs have TH activities10-100times greater than those offish AFPs and other kinds of AFPs. Antifreeze protein can be widely used in agriculture, medicine and food sciences.The testing, development and use of AFPs for various applications requires a large number of recombinant AFPs. However, due to the requirement for disulfide bond formation for the β-helix of insect AFPs, bacterial expression may result in inactive recombinant AFPs in the form of inclusion bodies. These proteins must then be denatured, refolded in the presence of guanidinium and then repurified to obtain active, correctly folded AFP. This procedure can only maintained some of the characteristics of protein. However, enough purified amounts of natural AFPs are difficult to be obtained due to the limited nature resources, which lead to the restriction of AFP application. Besides, the recombinant expression of beetle antifreeze protein in yeast system is difficult because of the numerous disulphide bonds in the protein. The objective of this study is to develop a high expression system for beetle antifreeze protein.Pichia pastoris has gained widespread attention as an expression system because of its potential to produce large quantities of heterologous proteins.Pichia offers many advantages over E. coli as an expression system.The promoter that is generally employed is the strong,tightly regulated, and inducible promoter of the AOX1, which has been characterized and incorporated into a series of P. pastoris expression vectors;the products of genes it directs can constitute up to30%of the total cell protein. The proteins produced in P. pastoris are typically subjected to appropriate posttranslational modifications such as proteolytic processing, folding, glycosylation, and the formation of disulfide bridges.Pichia pastoris yeast expression system has the ability of highly effective expression, high characteristics and so on stable, high secretion, high density growth. The character of recombinant product approach in the natural protein, not only the physical, but also the chemic, which are the prokaryotic expression system do not have. This article is to identify and establish a novel Pichia pastoris yeast expression system expression system for production of AFP.Microdera punctipennis is a special species in Tenebrionidae, Gurbantonggut Desert, Xinjiang, the northwest of China. Mpafp698, one of the antifreeze protein genes from Microdera Punctipennis.Expression of MpAFP698in Pichia pastoris.,and the recombinant High level expression of strains was screened, The optimization of fermentation conditions and application of the antifreeze protein from Microdera punctipennis, and measured the low-temperature protection activity, pH and thermal stability of the MpAFP698; the detection Mp AFP698to the organs of the mice, insect cell SF9and grapes low-temperature protective effect. The main findings are as follows:1. The recombinant expression plasmid pPIC9K-Mpafp698was constructed and transformed into E.coli Top10F'. The recombinant plasmid was transformed in to Pichia pastorris GS115.The transformants with multiple copies of insert were screened by DNA sequence analysis. The Mpafp698was identified by PCR in yeast, all the His+recombinant yeast was Mut+type; the production was identified and showed antigenicity.The selected transformants were fermented and induced by methanol in flasks. The protein expression was correctly detected by Tricine-SDS-PAGE and Western blotting.2. The recombinant strains was screened by G418resistance, express the protein and analyzed the orrelation between concentration and THA, the Recombinant frost the protein MpAFP698acid and alkali resistance and heat resistance, simultaneous determination of endogenous expression of the cold tolerance of yeast MpAFP698andof exogenous Add MpAFP698protective activity of bacteria. His+positive clones were screened on MD plates to the positive transformants by G418resistance selection, the high copy; the protein MpAFP698of insect antifreeze has a strong thermal stability characteristics, can tolerate the high temperature of100℃and can maintain a10-15min which activity changed little. MpAFP698with a strong acid-base stability characteristics, and can withstand extreme pH environment. The cold tolerance of yeast expression MpAFP698significantly increased; MpAFP698can significantly improve the survival rate of bacteria in the-7℃and42℃3.On the Fermentation of methanol induction concentration, induction time and temperature conditions to be optimized. Studies have shown that in the concentration of methanol was1.0%, temperature was25℃, the fermentation time is4d when MpAFP698expression.4.Detection of low temperatures, a small chest turtle shell antifreeze protein on the cell morphology of the organs of the mice, blood cell hemolysis, insect SF9cell survival, and cryopreservation of grapes, that a small chest turtle shell antifreeze protein can be significantly improved freezing small rat liver cell morphology, significantly reduced blood cell hemolysis at4℃and to improve the SF9cells after freeze-thaw survival rate; can significantly improve the water retention of different grape strains stored at4℃. In this study, a small chest turtle antifreeze protein large-scale eukaryotic fermentation provides a reference, as well as laid a theoretical foundation for further revealed the active mechanism of insect antifreeze protein antifreeze protection.