Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli.
Protein Expr Purif
; 35(1): 62-8, 2004 May.
Article
em En
| MEDLINE
| ID: mdl-15039067
ABSTRACT
The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endopeptidases
/
Proteínas Recombinantes de Fusão
/
Proteínas de Transporte
/
Vírus da Leucemia Murina
/
Escherichia coli
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2004
Tipo de documento:
Article
País de afiliação:
Hungria