title: A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli.
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.
authors.affiliation: College of Horticulture, Nanjing Agricultural University, 210095, Nanjing, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Weiguo
authors.initials: W
authors.lastname: Xu
authors.forename: Qiming
authors.initials: Q
authors.lastname: Sun