Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 490, Issue 1, Pages 8-16Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2017.05.153
Keywords
Recombinant DNA; Precision tagging; Type IIS restriction enzyme; Non-palindromic; THSD1
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Funding
- US National Institutes of Health [R03NS087416]
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Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs. (C) 2017 The Authors. Published by Elsevier Inc.
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