Splicing protein

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281. 

Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115. 

Evans, T.C., Benneg J., and Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Set. 7, 2256-2264. 

Fig. 1.3 The currently accepted chemical mechanism of protein splicing. 1 N-S(O) acyl shift, 2 transesterification, 3 cleavage by succinimide formation, 4 S(0)-N acyl shift.

Fig. 1.5 A Natural protein splicing, B Trans splicing with a split intein. The two fragments can be prepared separately and reassembled in vitro to form an active intein domain for protein ligation. C Central segment labeling using two different split inteins.

Residues involved in the protein splicing reaction are shown by a single character code of amino acids. N- and C-termini are indicated by NH2 and COOH respectively. 

A full-length human csk DNA that codes for Csk, a 50-kDa protein that catalyzes the phosphorylation of a tyrosine within the C-terminal tail of Srk, was inserted in a plasmid and in-frame with a modified intein-chitin binding domain (CBD) encoding sequence, where intein is a protein-splicing element. 130 The expressed protein underwent a conversion into a thioester of the N-terminal cysteine of the intein, but the normal second step of an intein splicing did not occur. The protein was then bound to a chitin resin and washed. The resin containing the bound protein was treated with 2% benzenethiol to cleave the thioester and give the free phenyl thioester, which was immediately treated with one of the synthetic... 

Native chemical ligation, which is described in Section 5.3, has proven to be a generally useful route to the total synthesis of proteins of up to medium size. A recent adaptation which utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein provides an interesting variant of this technique. 4,5 ... 

Figure 1.16 RNA splicing (a) and protein splicing (b). [Based on Y. Shao and S. B. H. Kent, Chemistry Biology 4,187 (1997).]...

Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990). Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H (+)-adenosine triphosphatase. Science 250, 651-657. 

Polypyrimidine tract-binding protein, a repressive regulator of protein splicing also pulmonary tuberculosis Protein transduction domain... 

Direct protein sequencing, by the methods discussed above (Sects. 5.1.5 and 5.1.6), is the preferred route to primary structure. It is now widely accepted that an indirect approach, via the sequence of cloned cDNA is also appropriate, and possibly easier. It does need to be established, however, that the protein primary structure is not affected by tissue specific mRNA processing (splicing or editing) or by post-translational modification such as N- or C-terminal processing or protein splicing. 

Key Words Expressed protein ligation (EPL) intein protein engineering protein splicing purification tag. 

Paulus, H. (2000) Protein splicing and related forms of protein autoprocessing. Annu. Rev. Biochem. 69, 447—496. 

Chong, S., Mersha, F. B., Comb, D. G., et al. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281. 

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