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

Abstract

The discovery of inteins in the early 1990s 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 trans-cleaving 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 co-workers (89) in this series. 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 PurificationOne 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). In these applications, a modified intein is expressed in fusion to an affinity tag and target protein, and once the fusion is affinity-purified, the intein is induced to cleave the target protein from the intein and tag (Fig. 1). The first commercial intein system was released by New England Biolabs in 1997 and employed a modified Sce VMA1 3 intein that is triggered to cleave at its N terminus (IMPACT system), or both N and C termini (IMPACT-CN system), by the addition of thiol compounds (e.g. Refs. 4 -7) (Fig. 1A, left). Shortly thereafter, inteins with rapid C-terminal cleaving activity were published. These inteins exhibit suppressed cleavage activity at pH 8.5, allowing purification of the tagged target, and are then induced to cleave by a shift to pH 6.5. The cleavage activity of these inteins is als...

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

“…To determine a maximum rate of cholesterolysis, we fit reaction progress curves at saturating cholesterol (350 µM) to a first order exponential, yielding an apparent k cat value of 0.001 sec −1 (t ½ ~ 11 minutes) ( Supporting Figure 3B ). This value at pH 7.1, the prevailing pH of the ER [25], is remarkably similar to the apparent rate of Hh precursor processing measured by pulse-chase experiments in cultured mammalian cells (0.0006 sec −1 ) [27]; it is also within range of autocatalytic “protein splicing” reactions brought about by inteins [22, 28-30], which may be ancestral to Hh proteins [14, 31]. Thus, the optical format facilitates kinetic analysis, and affords results that are reproducible and appear physiologically relevant.…”

Dynamics differentiate between active and inactive inteins

“…Inteins belong to the hedgehog intein superfamily of autoprocessing domains [2]. Due to the post-translational editing function, inteins have been used as powerful proteins in protein engineering, labeling, purification and control of protein function [3-10]. Inteins can also be used in drug design, as they are capable of undergoing selective activation of a protein, drug, or drug encapsulation in a viral coat [11].…”

Sequential formation of two branched intermediates during protein splicing of class three inteins

“…We conclude that there is no competition between the two catalytic cysteines for attack on the N-terminal splice junction and that the Bvi IcmO intein is unable to splice by the class 2 mechanism. The Bvi IcmO intein expands the repertoire of potential insertion sites for class 3 inteins in target proteins to include Cys for the numerous in vivo and in vitro applications based on intein technology (Aranko et al 2014; Topilina and Mills 2014; Wood and Camarero 2014). Inteins continue to prove to be intriguing and robust single turnover enzymes.…”