A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli.
Biotechnol Lett
; 31(7): 1051-7, 2009 Jul.
Article
en En
| MEDLINE
| ID: mdl-19330487
ABSTRACT
A novel protocol for producing recombinant Moloney murine leukemia virus (MMLV-RT) in Escherichia coli is reported. The optimized coding sequence for mature MMLV-RT was cloned into pET28a and over-expressed as an N-terminal His6-tagged fusion protein. An enterokinase (EK) recognition site was introduced between the His6-tag and MMLV-RT to release tag-free enzyme. Optimal expression of soluble His6-MMLV-RT was achieved by chaperone co-expression and lower temperature fermentation. The His6-tagged enzyme was first purified by Ni(2+) affinity chromatography. The bound enzyme was then eluted by EK digestion and the eluate was purified on an anion-exchange Q column to remove DNA and EK. Twenty-one milligram MMLV-RT was obtained from 1 l of bacterial culture.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Virales
/
ADN Polimerasa Dirigida por ARN
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Escherichia coli
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Virus de la Leucemia Murina de Moloney
Idioma:
En
Revista:
Biotechnol Lett
Año:
2009
Tipo del documento:
Article