Affinity Purification Of Wheat Xylanase Inhibiting Protein And Cloning And Expression Of Inhibiting Protein

Xylanase inhibit protein inhibit xylanase catalytic hydrolysis of xylan,bring the inconvenience to the application of xylanase in the industrial production,in addition to it also play important roles in the process in plant defense against pathogenic bacteria and pests.inhibiting protein XIP and chitinase has certain relations on evolution,and some of the chitinase without activity of chitinase and xylanase inhibitory.This experiment according to the enzyme-inhibiting protein interaction,adopt the method of immobilized xylanase affinity purification xylanase inhibit protein from wheat varieties XiaoYan 22 grain crude protein leaching solution,and from small suppress a total DNA of 22 get a gene cloning,in the e.coli and pichia expression of heterologous expression in the system,and analyze the heterologous expression product,and get a clone from total DNA of XiaoYan22 genes,heterologous expression in the E.coli and Pichia pastorios,and analyze the heterologous expression product.The results are as follows:(1)3%glutaraldehyde crosslinking for 16 h,immobilized enzyme ball recovery rate of 37.8%,immobilized enzyme affinity adsorption 48 h the adsorption rate was 47.88%of immobilized xylanase affinity adsorption xylanase inhibiting protein at 48 hours.The inhibition rate was 60.65%of affinity purified protein solution of xylanase inhibiting protein,the optimum inhibiting temperature of 30?,optimum restrain pH of 7.0.(2)According to the early stage of the experimental group get wild type xylanase inhibiting protein,it n-terminal sequence is SVSSVVS,sequence alignment,design primers,to extract the total DNA of wheat,PCR amplification get size suitable nucleic acids fragments,TA clone,plasmid extraction,plasmid sequences.By comparison with the sequence,choose gene of higher probability and gene sequence is identical with AY973230.1 and it has highly homologous with chitinase gene,selecte it as the object of study.(3)The purpose gene induced 5 hours in E.coli in 28?,collecting bacteria and ultrasonic broken,by SDS-PAGE detection,In broken supernatant did not find the size appropriated protein,in the broken precipitate found protein of size appropriate,presumably induced proteins to form the insoluble inclusion body,change induced conditions,make the purpose gene induced in E.coli at 16? for the night,by SDS-PAGE detection,in broken supernatant found the size appropriated soluble protein,The concentration of the purified protein is 770?g/ml,But the activity determination of protein find it without xylanase inhibitory activities,and almost no chitinase activity.(4)The gene induced with 0.5%methanol in Pichia pastorios in 28?and appeared induction target protein after 12 hours,after 84 hours,the quantity of objective protein is about 19.52mg/ml in pastorios but the protein was still less than xylanase inhibitory activity and chitinase activity.(5)Purpose gene induced protein structurally belong to chitinase,only without the chitinase activity,it conforms to the initial forecast,this study but without xylanase inhibitory activities,and thus didn't get the results of this study is the initial forecast.(6)The protein molecules of Prokaryotic induced expression of protein and eukaryotic induced expression of protein are same size,A little less than 29.0kDa.The protein molecules weight is 26.017kDa by software calculation,and according to the mobility of molecular weight is 28.79kDa,they are no difference.