Expression and purification of recombinant wheat translation initiation factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5.
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 24mg 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.
Mayberry
Laura K
LK
Department of Chemistry and Biochemistry and the Institute for Cell and Molecular Biology, University of Texas at Austin, USA.
Dennis
Michael D
MD
Leah Allen
M
M
Ruud Nitka
Kelley
K
Murphy
Patricia A
PA
Campbell
Lara
L
Browning
Karen S
KS
D000818:Animals; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014908:Triticum / Q000737:chemistry / Q000235:genetics
D039642:Eukaryotic Initiation Factors; D010940:Plant Proteins; D011994:Recombinant Proteins