期刊
MOLECULES
卷 26, 期 19, 页码 -出版社
MDPI
DOI: 10.3390/molecules26195948
关键词
halophilic intein; NMR; protein splicing; conditional protein splicing; C-terminal amidation; isotopic labeling; peptide purification; purification tag; self-cleaving tag
资金
- TEKES [1311/31/2014]
- Academy of Finland [131413, 137995, 277335]
- TaNeDS Europe 2017 grant program
- Novo Nordisk Foundation [NNF17OC0027550]
- Proteomics Unit at the Institute of Biotechnology
- Academy of Finland (AKA) [131413, 131413] Funding Source: Academy of Finland (AKA)
This study introduces an IIST system based on salt-inducible conditional protein splicing using the intein from Halorhabdus utahensis for purification of small peptides. It also describes a method for amidation and N-15 labeling of a single domain antibody using the same system.
An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT (TM) system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT (TM) system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated N-15-labeling of the C-terminal amide group of a single domain antibody (V-HH).
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