Expression and purification of SP6 RNA polymerase for in vitro transcription
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Updated: Nov 3, 2026

Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
Sergey A Zozulya1, Sergey A Vishnivetskiy2, Vsevolod V Gurevich2
1Enamine Ltd. (www.enamine.net), Kyiv, Ukraine.
Abstract:
Construction of a plasmid for efficient overexpression of SP6 RNA polymerase in E. coli and method of purification of multi-milligram quantities of this enzyme by two chromatographic steps is described. SP6 RNA polymerase purified by this method is highly active and stable in described storage conditions at -20 °C for many years. Due to its specific 5' sequence (5'-GAATACA…) uncapped mRNA synthesized by SP6 RNA polymerase can be used for cell-free translation yielding several times more protein product than uncapped mRNAs produced by other polymerases. These factors greatly reduce the costs of in vitro production of mRNA for cell-free translation, improving the feasibility of this protein expression system for various applications.
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