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Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody−intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

Elongation factor Tu from Thermus thermophiluscontaining six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography. Preformed aminoacyl-tRNA·EF-Tu(CHis6)·GTP ternary complexes were immobilized on Ni2+-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP. Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu′s affinity for aminoacyl-tRNA. The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts.

Suzanna Nock reviews an eye-opening e-discussion about women's relationships with midwives. [ABSTRACT FROM AUTHOR]