Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli

TRABUCCHI, ALDANA; GUERRA, LUCIANO L.; FACCINETTI, NATALIA I.; IACONO, RUBÉN F.; POSKUS, EDGARDO; VALDEZ, SILVINA N.

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2012 vol. 94 p. 1565 - 1576

0175-7598

Native proinsulin (PI) belongs to the class of thedifficult-to-express proteins in Escherichia coli. Problemsmainly arise due to its high proteolytic decay and troublesto reproduce the native disulphide pattern. In the presentstudy, human PI was produced in E. coli as a fusionthioredoxin protein (Trx-PI). Such chimeric protein wasobtained from the intracellular soluble fraction, and it waspurified in one step by affinity chromatography onimmobilized phenylarsine oxide. Trx-PI was also recoveredfrom inclusion bodies and purified by anion exchangechromatography. The product identity and integrity wereverified by mass analysis (22,173.5 Da) and mapping withStaphylococcus aureus V8 protease. Native PI folding wasevaluated by biochemical and also by immunochemicalanalysis using specific sera from PI antibody-positivediabetic patients that recognise conformational discontinueepitopes. Dose?response curves showed identity betweenstandard PI and Trx-PI. Moreover, surface plasmon resonancetechnique verified the correct conformation of therecombinant protein. The biochemical and immunochemicalassays demonstrated the integrity of the chimera and theepitopes involved in the interaction with antibodies. Inconclusion, it was possible to obtain with high-yieldpurified human PI as a fusion protein in E. coli and usefulfor analytical purposes.