Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5

Laura K. Mayberry, Michael Dennis, M. Leah Allen, Kelley Ruud Nitka, Patricia A. Murphy, Lara Campbell, Karen S. Browning

Research output: Chapter in Book/Report/Conference proceedingChapter

25 Citations (Scopus)

Abstract

Protein synthesis initiation factors from wheat germ were cloned into E. coli expression vectors for expression and purification. The ability to obtain large amounts of functional initiation factors and mutants of the factors will facilitate the biophysical and biochemical analysis of the process of initiation in plants. The initiation factors, eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5, were successfully expressed and purified from E. coli. In most cases, the use of 6X-histidine tags was avoided to prevent any possible artifacts of folding or activity because of the presence of the tag. The amounts of highly purified wheat initiation factors obtained ranged from 0.5 to 24 mg of protein per liter of culture, depending on the particular initiation factor. The initiation factors were of very high purity, and the activities of the wheat recombinant factors purified from E. coli were found to be comparable to or better than those purified from wheat germ.

Original languageEnglish (US)
Title of host publicationTranslation Initiation
Subtitle of host publicationReconstituted Systems and Biophysical Methods
PublisherAcademic Press Inc.
Pages397-408
Number of pages12
ISBN (Print)9780123739698
DOIs
StatePublished - Jan 1 2007

Publication series

NameMethods in Enzymology
Volume430
ISSN (Print)0076-6879

Fingerprint

Peptide Initiation Factors
Triticum
Purification
Escherichia coli
Biochemical Phenomena
Histidine
Artifacts
eIF-4B
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Mayberry, L. K., Dennis, M., Leah Allen, M., Ruud Nitka, K., Murphy, P. A., Campbell, L., & Browning, K. S. (2007). Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. In Translation Initiation: Reconstituted Systems and Biophysical Methods (pp. 397-408). (Methods in Enzymology; Vol. 430). Academic Press Inc.. https://doi.org/10.1016/S0076-6879(07)30015-3
Mayberry, Laura K. ; Dennis, Michael ; Leah Allen, M. ; Ruud Nitka, Kelley ; Murphy, Patricia A. ; Campbell, Lara ; Browning, Karen S. / Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. Translation Initiation: Reconstituted Systems and Biophysical Methods. Academic Press Inc., 2007. pp. 397-408 (Methods in Enzymology).
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abstract = "Protein synthesis initiation factors from wheat germ were cloned into E. coli expression vectors for expression and purification. The ability to obtain large amounts of functional initiation factors and mutants of the factors will facilitate the biophysical and biochemical analysis of the process of initiation in plants. The initiation factors, eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5, were successfully expressed and purified from E. coli. In most cases, the use of 6X-histidine tags was avoided to prevent any possible artifacts of folding or activity because of the presence of the tag. The amounts of highly purified wheat initiation factors obtained ranged from 0.5 to 24 mg of protein per liter of culture, depending on the particular initiation factor. The initiation factors were of very high purity, and the activities of the wheat recombinant factors purified from E. coli were found to be comparable to or better than those purified from wheat germ.",
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Mayberry, LK, Dennis, M, Leah Allen, M, Ruud Nitka, K, Murphy, PA, Campbell, L & Browning, KS 2007, Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. in Translation Initiation: Reconstituted Systems and Biophysical Methods. Methods in Enzymology, vol. 430, Academic Press Inc., pp. 397-408. https://doi.org/10.1016/S0076-6879(07)30015-3

Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. / Mayberry, Laura K.; Dennis, Michael; Leah Allen, M.; Ruud Nitka, Kelley; Murphy, Patricia A.; Campbell, Lara; Browning, Karen S.

Translation Initiation: Reconstituted Systems and Biophysical Methods. Academic Press Inc., 2007. p. 397-408 (Methods in Enzymology; Vol. 430).

Research output: Chapter in Book/Report/Conference proceedingChapter

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PB - Academic Press Inc.

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Mayberry LK, Dennis M, Leah Allen M, Ruud Nitka K, Murphy PA, Campbell L et al. Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. In Translation Initiation: Reconstituted Systems and Biophysical Methods. Academic Press Inc. 2007. p. 397-408. (Methods in Enzymology). https://doi.org/10.1016/S0076-6879(07)30015-3