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

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

Research output: Chapter in Book/Report/Conference proceedingChapter

27 Scopus citations

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

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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