P53C Protein Expression By Recombinant Bacteria And Chromatographic Purification

p53protein is one of the important cancer suppressers in humans and a necessarytranscription factor under stress. In recent years, it is a research hotspot to express p53protein with genetic engineering bacteria and establish relevant purification methods.First of all, a genetic engineering bacteria was constructed to express the targetprotein. p53core domain (p53C) is residues94-312of p53, which covers the wholeDNA binding domain. More than85%missense mutations can be mapped to thishighly conservative region. Therefore the encoding gene of p53C was inserted into thepolylinker region of pRSET(A). The ligated plasmid was transformed into Escherichiacoli strain BL21(DE3) to build a recombinant bacteria which could heterogeneouslyexpress p53C protein. The bacterial culture conditions, including induction startingtime, induction time and inducer concentration, were optimized and the experimentalresults indicated that high-efficient target protein expression was achieved when theinduction was performed in early logarithmic phase of bacterial growth, with aninduction time of8h and an inducer concentration of1mmol/L. Thereafter,SP-Sepharose cation-exchange chromatography was utilized for the purification.Three elution strategies were examined, including isocratic elution, linear gradientelution and step gradient elution. Step gradient elution with a Nacl concentration of80%was proven as the best operating condition and the purity of target protein couldreach21.2%. pH was also found to affect the separation performance. The highestyield of p53C protein was observed in pH8.0, where the purity was21.8%. In addition,preliminary results on the separation of p53C using gel filtration chromatography andaffinity chromatography were reported. However, the results were not satisfactory.This research has established a high-efficient protocol for the expression andpurification of p53C protein. It would be helpful for the modification of p53C proteinand the development of anti-cancer drug.