INVESTIGADORES
POSKUS Edgardo
artículos
Título:
Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli
Autor/es:
TRABUCCHI A, GUERRA LL, FACCINETTI, NI, IACONO RF, POSKUS E, VALDEZ SN
Revista:
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Editorial:
SPRINGER
Referencias:
Lugar: Berlin; Año: 2012 p. 1 - 12
ISSN:
0175-7598
Resumen:
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. Doseresponse 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.