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

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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 Ni2+ 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.

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Acknowledgements

The authors are grateful to Dr. Hideki Yanagi for kindly providing the pG-Tf2 plasmid. This study is supported by International Technology Cooperation Division of Jiangsu Province and the government of Xuzhou City.

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  1. Qiming Sun

    Present address: University of California, Berkeley, CA, USA

Authors and Affiliations

  1. College of Horticulture, Nanjing Agricultural University, 210095, Nanjing, People’s Republic of China

    Yu Chen

  2. ProbeGene Biotechnology Corporation, 210029, Nanjing, People’s Republic of China

    Weiguo Xu & Qiming Sun

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  1. Yu Chen
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  2. Weiguo Xu
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  3. Qiming Sun
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Correspondence to Qiming Sun.

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Chen, Y., Xu, W. & Sun, Q. 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, 1051–1057 (2009). https://doi.org/10.1007/s10529-009-9977-5

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  • DOI: https://doi.org/10.1007/s10529-009-9977-5

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