A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli

作者：Chen Yu; Xu Weiguo; Sun Qiming^*

来源：Biotechnology Letters, 2009, 31(7): 1051-1057.

DOI：10.1007/s10529-009-9977-5

• Chaperone
• Enterokinase
• Leukemia virus
• Lower temperature
• Reverse transcriptase

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.

• 出版日期2009-7
• 单位南京农业大学