Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli
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Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose–response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.
KeywordsThioredoxin proinsulin Protein expression Immunoreactivity Diabetes mellitus Autoantibodies
We thank C. Mazza and A.G. Krochik at the J. P. Garrahan National Pediatrics Hospital and the Hemotherapy Division at the José de San Martín Clinical Hospital (Buenos Aires, Argentina) for collecting and providing the sera of diabetic patients and control individuals. We are grateful to Graeme Bell, University of Chicago, USA, for the gift of pBR328–preproinsulin vector. We also thank Eli Lilly for the generous supply of standard proinsulin. This work was supported in part by grants from FONCYT Programme of the Agency for Science and Technology Promotion (ANPCyT), National Research Council (CONICET) and the University of Buenos Aires, Buenos Aires, Argentina.
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