Intein‐mediated protein ligation: Harnessing nature's escape artists

Abstract

Inteins are naturally occurring proteins that are involved in the precise cleavage and formation of peptide bonds in a process known as protein splicing. Genetic engineering has allowed the controllable cleavage of peptide bonds at either the N‐ or C‐terminus of the intein. Inteins displaying controllable cleavage have been used in the isolation of bacterially expressed proteins possessing either a C‐terminal thioester or an N‐terminal cysteine. The specific placement of these reactive groups has allowed either protein–protein or protein–peptide condensation through a native peptide bond. This review describes the methods used to specifically generate these reactive groups on bacterially expressed proteins and some applications of this technique, known as intein‐mediated protein ligation. Furthermore, a versatile two intein (TWIN) system will be described which enables the circularization and polymerization of bacterially expressed proteins or peptides. © 2000 John Wiley & Sons, Inc. Biopoly 51: 333–342, 1999

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…In general, this affinity tag is combined in tandem with self-splicing inteins [142][143][144] and is commercially available via the IMPACT™ (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system. A chitin matrix is employed for affinity purification of the recombinant protein, whereas self-cleavage of the thioester bond is induced by a thiol reagent (e.g., 1,4-dithiothreitol or β-mercaptoethanol) or pH adjustment combined with a temperature shift [144,145]. To reduce non-specific binding, either nonionic detergents or high salt concentrations are used.…”

Small Ubiquitin-like Modifier (SUMO) Modification of E1 Cys Domain Inhibits E1 Cys Domain Enzymatic Activity

“…Conversion of thioester bonds to peptide bonds is not unique to ubiquitin-like modifications, but is used by other cellular peptide synthesis processes, such as intein-mediated protein ligation (23) and nonribosomal peptide synthesis (24). Ubiquitin-like modifications are unique in that an additional transthioesterification step is required: transfer of SUMO from the E1⅐Ubl thioester to the E2⅐Ubl thioester, which is responsible for substrate recruitment (25).…”

Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins

“…Classical chemical methods are not able to easily reproduce such modifications [85]. Inteins, in contrast, provide a cheaper and more versatile approach to generate cyclic peptides and proteins directly in a host organism using a process termed intein-mediated protein ligation (IPL) [36] [87]. In this process the protein of interest (i.e.…”

“…The C-terminal activated thioes-ter of the target is now able to react with the N-terminal cysteine leading to the desired cyclization [88]. This strategy forms the basis of a commercially available kit that uses the vector pTWIN of the TWointein system, which contains the two required inteins [87]; the mutated version of the intein DnaB from Synechocystis sp PCC6803 is linked to the C-terminal end of the target protein, while an intein from Mycobacterium xenopy, MXE GyrA, is linked to its N-terminus. However, this approach has major drawbacks largely due to the low cleavage efficien- Figure 6.…”

“…The Sec modification is subsequently triggered by the activation of splicing, in a process whereby intein activity catalyzes the formation of a link between the target protein and the Sec-containing module [104]. A more efficient and rapid method has recently been developed and patented by the name of Sectein [87]. In this system the expression of the selenoprotein exploits the recognition of the UGA codon by the SECIS element and the splicing reaction induced by the intein (Figure 7).…”