Studies On Expression And Puirifcation Of Recombinant Human PDX-1in Pichia Pastoris

Diabetes，is a group of metabolic diseases in which a person has high blood sugar，eitherbecause the pancreas does not produce enough insulin，or because cells do not respond to theinsulin that is produced. Insulin is the one and only hormone produced by organism whichcould reduce the blood sugar level. As an indispensable transcription factor，PDX-1playimportant roles in the growing，development，differentiation of pancreas，and the secretionof insulin. In recent years，due to the continuous development of fundamental research，thestudy of this protein grows deeper. Hence，the production of PDX-1using genetic engineeringhas profound implications.This study is focus on the following points:1、The construction of Pichia pastoris engineering strainThe recombinant expression vector was linearized，and transformed into P. pastoris X-33by electroporation. The gene encoding TAT-PDX-1was integrated into the genomic DNA andwhich was confirmed by PCR.The results of experiments showed the TAT-PDX-1Pichia pastoris expression system couldproduce TAT-PDX-1under the condition of methanol induce. There were positive bands in theSDS-PAGE and Western Blot analysis.2、The optimization of condition of yeast expression systemThe strains which could express TAT-PDX-1at high level were selected，and they wereinoculated into small amount of BMGY. The supernatant was analyzed by SDS-PAGE，andthe results showed the best fermentation time was72hours，and the optimized pH was5.5.3、The scale-up fermentation of yeast expression systemThe selected strain was inoculated into2L BMGY under the optimized condition. Theprotein in the supernatant was analyzed by SDS-PAGE，and the result showed the constantexpression level of TAT-PDX-1，which was consistent with the expectation.4、The purification of TAT-PDX-1The purification strategy was confirmed after several tries. The supernatant was obtained bycentrifugation，and it was processed by ultrafiltration and ion-exchange. Finally，the highlypurified protein was acquired.