Expression and purification of SP6 RNA polymerase for in vitro transcription

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.