Intein Applications Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods

The discovery of inteins in the early 1990’s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and transcleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization and protein purification. The ability of inteins to form and cleave specific peptide bonds in a variety of contexts has enabled the development of powerful new tools in molecular biology. Initial applications focused on self-cleaving affinity tags and protein modification and labeling, and utilized mutations that altered the native splicing reactions described by Perler and Mills in this issue. An important advantage of intein-based methods is that they are generally enzymatic in nature, and therefore exhibit highly specific activities under physiological conditions. Although thiol compounds are used in some applications, the chemistries involved in most intein methods are innocuous to their various target proteins. Intein-based methods have greatly expanded in subsequent years through the discovery of hundreds of additional inteins, and new applications in protein activity regulation and modification have followed. An important underlying theme in this work has been the discovery and development of highly efficient split inteins for advanced applications in vitro and in vivo. These trans-splicing and cleaving inteins have been employed for applications ranging from the purification of recombinant products to the in vivo control of protein function and labeling. PROTEIN PURIFICATION One of the first major applications of inteins was the development of self-cleaving affinity tags for the recovery of untagged target proteins in recombinant expression systems (1-3). http://www.jbc.org/cgi/doi/10.1074/jbc.R114.552653 The latest version is at JBC Papers in Press. Published on April 2, 2014 as Manuscript R114.552653 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on July 2, 2017 hp://w w w .jb.org/ D ow nladed from