Expression and purification of the alpha-subunit of eukaryotic initiation factor eIF2: use as a kinase substrate

Protein Expression and Purification
S R KimballL S Jefferson

Abstract

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regul...Continue Reading

References

Jul 31, 1991·Biochemical and Biophysical Research Communications·H Mellor, C G Proud
Jun 1, 1995·Trends in Biochemical Sciences·C G Proud
Jan 13, 1994·Biochimica Et Biophysica Acta·N T Redpath, C G Proud
Mar 15, 1996·European Journal of Biochemistry·V M Pain
Apr 1, 1997·International Journal of Hematology·C E SamuelH Tanaka

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Citations

Jan 24, 2002·American Journal of Physiology. Gastrointestinal and Liver Physiology·Maria Dolors SansJohn A Williams
Apr 27, 1999·The International Journal of Biochemistry & Cell Biology·L S JeffersonS R Kimball
Jun 11, 2003·The Journal of Experimental Biology·Julian L PakayMichael Guppy
Nov 18, 2005·Protein Expression and Purification·Rajasekhar N V S SuraganiKolluru V A Ramaiah

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