The Fermentation And Renaturation Of Kallikrein

Kallikrein(KLK)existing in all parts of human is an important partof KKS system used to control agglomeration of plaque, improve bloodcircle, and reduce blood pressure, can cure diabetes, mellitushypertension and etc. My experiment focus on the FermentationCondition, denaturation and renaturation techniques of recombinanthuman kallikrein optimization.Optimize the factors that influence the results of fermentation yieldof KLK from my laboratory. Finally get the optimum fermentationcondition:inducers concentration,0.6mmol L^-1; time of induction,4hour;the concentration ofAmp,60mg L^-1; the temperature of cultivation,37℃.After optimization, induce the recombinant KLK engineeredmicroorganism with IPTG, and the targeted protein existed in the style ofinclusion body. Release inclusion body with ultrasonication. Throughthree times of washing with buffer, the protein concentration reach71.93%; dissolve the includion body with8M Urea, and the proteinconcentration reaches1731μg mL^-1in saturation state.In the method of dilution ranaturation,12hours later, the finalconcentration reach181.51μg mL^-1,64.23μg mL^-1,39.14μg mL^-1,respectively, and enzyme activity reach0.85U mg^-1,7.68U mg^-1,11.24U mg^-1respectively. Enzyme activity is higher within theconcentration of50μg mL^-1. In the methods of single-step dialysis ranaturation and multiple-stepdialysis renaturation, the final protein concentration of KLK reach84.52μg mL^-1and75.42μg mL^-1respectively and enzyme activity reach0.63U mg^-1,3.13U mg^-1respectivelyColumn chromatography was choosed as the method of renaturation.With the method of SEC (Sephadex-G75), the procedure cannot go onbecause of blocking and instability of ilution. With the method of Ureagradient of SEC, optimize the length of urea gradient from10%to20%to30%. Protein recovery rate reached64.27%,75.68%,71.26%. Specificactivity reached23.21U mg^-1,48.61U mg^-1,38.27U mg^-1respectively.With the optimum gradient length of20%, the activity of KLK reach48.61U mg^-1higher than dilution method and dialysis ranaturationmethod; With the method of IEC, protein recovery rate gets56.23%,while IEC can't make includion body renatured.With the method of traditional renaturation and columnchromatography, the inclusion body of KLK can get renatured to someextent. Because of better effect and shorter time, sec(Sephadex-G75) withurea gradient could be choosed as an effective method for renaturation ofKLK.