Abstract
Preparations of rHMfA (recombinant histone A from M ethanothermus f ervidus) synthesized in E. coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMf A molecules, ∼40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), ∼50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only ∼10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMf A molecules synthesized in Mt. fervidus. Expression of the hmfA gene in E. coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMf A preparations in which >85% of the molecules have the fully processed, native N-terminal sequence. This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E. coli.
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Sandman, K., Grayling, R. & Reeve, J. Improved N-terminal Processing of Recombinant Proteins Synthesized in Escherichia coli. Nat Biotechnol 13, 504–506 (1995). https://doi.org/10.1038/nbt0595-504
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DOI: https://doi.org/10.1038/nbt0595-504
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