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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
References
Sherman, F., Stewart, J.W. and Tsunasawa, S. 1985. Methionine or not methionine at the beginning of a protein. BioEssays 3: 27–31.
Adams, J.M. 1968. On the release of the formyl group from nascent proteins. J. Mol. Biol. 33: 571–589.
Mazel, D., Pochet, S. and Marlière, P. 1994. Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13: 914–923.
Sandman, K., Grayling, R.A., Dobrinski, B., Lurz, R. and Reeve, J.N. 1994. Growth phase dependent synthesis of histones in the archaeon Methanothermus fervidus. Proc. Natl. Acad. Sci. USA 91: 12624–12628.
Zillig, W., Palm, P., Reiter, W-D., Gropp, F., Püller, G. and Klenk, H-P. 1988. Comparative evaluation of gene expression in archaebacteria. Eur. J. Biochem. 173: 473–482.
Sandman, K., Krzycki, J.A., Dobrinski, B., Lurz, R. and Reeve, J.N. 1990. HMf, a DNA-binding protein isolated from the hyperthermophilic archaeon Methanothermus fervidus, is most closely related to histones. Proc. Natl. Acad. Sci. USA 87: 5780–5791.
LeGendre, N., Mansfield, M., Weiss, A. and Matsudaira, P. 1993. Purification of proteins and peptides by SDS-PAGE, p. 71–101. In: Practical Guide to Protein and Peptide Purification for Microsequencing. Matsudaira, P. (Ed.). Academic Press, San Diego, CA.
Ben-Bassat, A., Bauer, K., Chang, S.-Y., Myambo, K., Boosman, A. and Chang, S. 1987. Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169: 751–757.
Tabassum, R., Sandman, K.M. and Reeve, J.N. 1992. HMt, a histonerelated protein from Methanobacterium thermoautotrophicum ΔH. J. Bacteriol. 174: 7890–7895.
Smith, B.J. 1984. Acetic acid-urea polyacrylamide gel electrophoresis of proteins, p. 63–73. In: Methods of Molecular Biology, Volume 1, Proteins. Walker, J. M. (Ed.). Humana Press, Clifton, NJ.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0595-504
This article is cited by
-
Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation
Journal of Industrial Microbiology and Biotechnology (2012)