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Self‐cleaving purification tags re‐engineered for rapid Topo® cloning
Author(s) -
Wu WanYi,
Gillies Alison R.,
Hsii Judy F.,
Contreras Lydia,
Oak Seachol,
Perl Michael B.,
Wood David W.
Publication year - 2010
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.430
Subject(s) - intein , cleavage (geology) , cloning (programming) , mutant , recognition sequence , dna , biochemistry , chemistry , computational biology , biology , microbiology and biotechnology , gene , combinatorial chemistry , rna , computer science , paleontology , restriction enzyme , fracture (geology) , rna splicing , programming language
In this work, our previously reported ΔI‐CM and ΔI G ‐CM mutant inteins were rationally re‐engineered to be compatible with Invitrogen's Topo® cloning system. The resulting new inteins include the vaccinia virus topoisomerase I DNA recognition sequence TCCTT at their 3′ ends, making them compatible with the highly convenient one‐step Topo® cloning method. Addition of the Topo® recognition sequence resulted in an altered amino acid sequence at the C‐termini of the inteins, changing their final five residues from VVVHN to VLVHN. Despite this change, these modified inteins retained their self‐cleaving function, and continue to exhibit pH and temperature‐sensitive cleaving characteristics as required for their use in generating self‐cleaving affinity tags. Although the C‐terminal modification decreased the intein cleavage rate under optimal conditions, cleavage can typically be completed within several hours at pH 6.5 and 37°C. In particular, the modified ΔI GT ‐CM intein is compatible with both the Topo® and Gateway® methods simultaneously, allowing fast parallel construction of multiple expression vectors with varying combinations of target proteins, self‐cleaving affinity tags and promoters. These newly engineered inteins increase the functionality of intein‐mediated technology, making it possible to explore a large number of combinations between target genes, self‐cleaving affinity tags and expression hosts in a fast and efficient manner. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010