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Characteristics of protein splicing in trans mediated by a semisynthetic split intein
Author(s) -
Lew Belinda M.,
Mills Kenneth V.,
Paulus Henry
Publication year - 1999
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(1999)51:5<355::aid-bip5>3.0.co;2-m
Subject(s) - intein , chemistry , protein splicing , rna splicing , computational biology , biochemistry , gene , biology , rna
Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans‐splicing reaction mediated by a naturally expressed, approximately 100‐residue N‐terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C‐terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37°C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C‐terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N‐terminal fragment and either the naturally expressed 107‐residue C‐terminal portion of the intein, much smaller synthetic peptides, or the 107‐residue C‐terminal intein fragment modified by fusion of a maltose binding protein domain to its N‐terminus. The ability to use different types of the C‐terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation. © 2000 John Wiley & Sons, Inc. Biopoly 51: 355–362, 1999