PMID: 9634792May 1, 1995Paper

Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli

Bio/technology
K SandmanJ N Reeve

Abstract

Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E. coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA 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 rHMfA 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.

References

Dec 1, 1992·Journal of Bacteriology·R TabassumJ N Reeve
Aug 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·K SandmanJ N Reeve
Jul 1, 1985·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·F ShermanS Tsunasawa
May 2, 1988·European Journal of Biochemistry·W ZilligH P Klenk
May 14, 1968·Journal of Molecular Biology·J M Adams
Dec 20, 1994·Proceedings of the National Academy of Sciences of the United States of America·K SandmanJ N Reeve
Jan 1, 1984·Methods in Molecular Biology·B J Smith

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Citations

Nov 21, 2007·Nature Methods·Clemens AchmüllerBernhard Auer
May 10, 2012·Protein Expression and Purification·Minmin LiuSusan S Wallace
May 1, 1996·FEMS Microbiology Reviews·R A GraylingJ N Reeve
Jun 17, 1999·Journal of Molecular Biology·S L Pereira, J N Reeve
Oct 8, 1998·FEBS Letters·W AlbigD Doenecke
Dec 3, 2008·Journal of Bacteriology·Ulrike Friedrich-JahnHarald Huber
Apr 3, 2001·Applied and Environmental Microbiology·V EnoufG Corthier
Jun 12, 2002·The Journal of Biological Chemistry·Frederic MarcJohn N Reeve
Mar 8, 2000·Journal of Molecular Biology·D J SoaresJ N Reeve

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