Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification.

Autor: Warren TD; Johns Hopkins University, Department of Chemical and Biomolecular Engineering, 3400 North Charles Street, Baltimore, MD 21218, United States. Electronic address: twarre12@jhu.edu., Coolbaugh MJ, Wood DW
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2013 Oct; Vol. 91 (2), pp. 169-74. Date of Electronic Publication: 2013 Aug 19.
DOI: 10.1016/j.pep.2013.08.006
Abstrakt: The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.
(Copyright © 2013 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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