title: Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli.
abstract: 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.
authors.affiliation: School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), Junín 956, 4to piso (C1113AAD), Buenos Aires, Argentina.
authors.forename: Aldana
authors.initials: A
authors.lastname: Trabucchi
authors.forename: Luciano Lucas
authors.initials: LL
authors.lastname: Guerra
authors.forename: Natalia Inés
authors.initials: NI
authors.lastname: Faccinetti
authors.forename: Ruben Francisco
authors.initials: RF
authors.lastname: Iacono
authors.forename: Edgardo
authors.initials: E
authors.lastname: Poskus
authors.forename: Silvina Noemí
authors.initials: SN
authors.lastname: Valdez
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D011384:Proinsulin