Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli


By jpeza - Posted on 11 Noviembre 2010

Fecha Publicación: 
1 Ene 1992
Nombre de Revista: 
Datos del paper
Autor Principal: 
Mark A.J. Taylor
Volumen: 
5
Issue: 
5
Página Inicial: 
455
Página Final: 
459
Abstract: 

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli.
This inactive precursor can subsequently be processed to yield active
enzyme. Sufficient protein can be produced using this
system for X-ray crystallographic structure studies
of engineered proteinases. A cDNA clone encoding propapain, a precursor
of the papaya proteinase, papain, was expressed in E.coli
using a T7 polymerase expression system. Insoluble recombinant protein
was solubilized in 6 M guanidine hydrochloride and
10 mM dithiothreitol, at pH 8.6. A
protein-glutathione mixed disulphide was formed by dilution into
oxidized glutathione and
6 M GuHCl, also at pH 8.6. Final refolding and
disulphide bond formation was induced by dilution into 3 mM cysteine at
pH
8.6. Renatured propapain was processed to active
papain at pH 4.0 in the presence of excess cysteine. Final processing
could
be inhibited by the specific cysteine proteinase
inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This
indicates
that final processing was due to a cysteine
proteinase and suggests that an autocatalytic event is required for
papain maturation.

Dirección del Autor: 

AFRC, Institute of Food Research Earley Gate, Whiteknights Road, Reading, RG6 2EF, UK

Keywords: 
cDNA E.coli papain precursor recombinant
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