期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 99, 期 19, 页码 8151-8161出版社
SPRINGER
DOI: 10.1007/s00253-015-6796-6
关键词
Protein labeling; Split intein; trans-splicing; Site-specific modifications
资金
- National Science and Engineering Research Council (NSERC) of Canada
- National Natural Science Foundation of China [31470836, 31070698]
- Shanghai Natural Science Foundation [13ZR1400600]
- Shanghai Science and Technology Commission of projects [14521100700, 14520720200]
- Ministry of Education of China [20120075110007, TS2011DHDX025]
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.
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