INVESTIGADORES
MENZELLA Hugo Gabriel
artículos
Título:
High recovery of prochymosin from inclusion bodies using controlled air oxidation
Autor/es:
MENZELLA H.G.; GRAMAJO H.; CECCARELLI E.
Revista:
PROTEIN EXPRESSION AND PURIFICATION
Editorial:
ACADEMIC PRESS INC ELSEVIER SCIENCE
Referencias:
Año: 2002 vol. 25 p. 248 - 255
ISSN:
1046-5928
Resumen:
Refolding of proteins from inclusion bodies is a field of increasing
interest for obtaining large amounts of active enzymes. Consequently,
the development of inexpensive and scalable processes is required. This
is particularly challenging in the case of eukaryotic proteins
containing cysteines, which may form disulfide bonds in the native
active protein. Previous studies have shown that the formation of
disulfide bonds is essential for the refolding of prochymosin. In this
work we demonstrate that air oxidation can be efficiently used for the
refolding of prochymosin and that 48% of the unfolded protein can be
recovered as active enzyme at a final protein concentration of 0.8
mg/ml. Refolding of the protein strictly correlates with the change in
pH of the refolding solution. We were able to follow the degree of
oxidative renaturation of the prochymosin by simply measuring pH. Thus,
the scaling up of the refolding system under controlled conditions was
easily achieved. Analyses of different substances as folding aids
indicate that the use of L-arginine or neutral surfactants improves the
recovery of active protein up to 67% of the initial protein. The overall
results indicate that prochymosin can be efficiently and inexpensively
refolded with high yields by controlled air oxidation.