期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 279, 期 20, 页码 20685-20691出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M400887200
关键词
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Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30degreesC and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.
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