title: Cysteine-free non-canonical C-intein for versatile protein C-terminal labeling through trans-splicing.
abstract: Site-specific protein labeling are powerful means of protein research and engineering; however, new and improved labeling methods are greatly needed. Split inteins catalyze a protein trans-splicing reaction that can be used for enzymatic and nearly seamless protein labeling. Non-canonical S11 split intein has been used in an earlier method of protein C-terminal labeling; however, its relatively large (~150 aa) N-intein fused to the target protein often hindered protein expression, folding, and solubility. To solve this problem, here, we have designed and demonstrated a new method of protein C-terminal labeling, by first engineering a functional non-canonical S1 split intein that has an extremely small (12 aa) N-intein and a cysteine-free C-intein. An engineered Rma DnaB S1 split intein was modified to have a cysteine-free C-intein, while still retaining its robust trans-splicing function, which permitted the C-extein in a C-precursor to have a single cysteine for easy and specific linkage with desired labeling groups. The resulting new and generally useful method has two unique advantages: (1) The extremely small (12 aa) N-intein, which must be fused to the C terminus of the target protein, is less likely to hinder the protein expression, folding, and solubility; and (2) the single cysteine in the C-extein may be readily linked to a variety of labeling or modification groups using commercially available reagents.
authors.affiliation: College of Textiles, Donghua University, Shanghai, 201620, China.
authors.forename: Xudong
authors.initials: X
authors.lastname: Dai
authors.forename: Qijing
authors.initials: Q
authors.lastname: Xun
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
authors.forename: Qing
authors.initials: Q
authors.lastname: Meng
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D007700:Kinetics; D008969:Molecular Sequence Data; D011486:Protein C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019154:Protein Splicing; D020040:Trans-Splicing
chemical_list: D011486:Protein C; D003545:Cysteine