In particular, fusions to inteins are, to varying extents, susceptible to premature extein cleavage, both and during initial purification from cell lysates, which reduces the isolated yield of the intein fusion needed for the subsequent thiolysis step.19 Importantly, the cleaved extein side-product is unreactive toward EPL, and its separation from the desired -thioester or the ligation product is often difficult for large proteins, such as antibodies.19 To compound matters, the thiolysis reaction itself can be slow and inefficient, further strengthening the need to develop customized purification regimes, involving multiple chromatographic actions, to isolate the desired product from complex mixtures.20?22 Collectively, these technical issues mean that a considerable investment in time and resources is usually required before a semisynthetic protein is obtained in useful quantities. To overcome the various drawbacks associated with the intein thiolysis process central to EPL, we envisioned an alternative strategy based on Tamsulosin hydrochloride naturally occurring split inteins. for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically altered monoclonal antibody. Introduction The chemical modification of proteins is an established tool for studying the structure, function, and regulation of this class of biopolymer.1 Moreover, in recent years, a great deal of effort has been directed toward the modification of proteins for therapeutic applications.2 Traditionally, protein conjugation chemistries have exploited the reactivity of surface-exposed nucleophilic amino acids, such as cysteine or lysine, however, these methods typically result in heterogeneous mixtures of products, which can complicate biological studies or efficacious medicinal applications. To address this concern, several strategies have been developed for the site-specific modification of proteins, ranging from total chemical synthesis (usually via native chemical ligation, NCL)3 to the Tamsulosin hydrochloride genetic incorporation of unnatural amino acids4 or bio-orthogonal functional groups.5 In between these two extremes lie a variety of semisynthetic approaches.5?7 The most widely used protein semisynthesis technique is an extension of NCL termed expressed protein ligation (EPL) in which a recombinant protein -thioester building block is ligated to a synthetic molecule equipped with a 1,2-aminothiol moiety (most commonly an N-terminal Cys-containing peptide) through the formation of a native peptide bond8,9 (Scheme 1). Since its inception, EPL has been applied to a wide variety of proteins, including enzymes,10 ion channels,11 transcription factors,12 transmembrane receptors,13 and antibodies14 (for reviews see refs (15 and 16)). One of the basic requirements of EPL is usually a thioester group at the C-terminus of a recombinant protein. This reactive handle is introduced by exploiting a process known as protein splicing (Scheme 1), which is usually mediated by an autoprocessing domain name called an intein.16,17 Protein splicing typically takes place through the formation of one or more protein thioester intermediates, which ultimately handle to form a native peptide bond between the sequences flanking the intein (referred to as N- and C-exteins). By using appropriate intein mutants, it is possible to intercept these intermediates with exogenous thiols, resulting in an N-extein of choice being cleaved from the mutant intein as a reactive -thioester derivative suitable for chemical Tamsulosin hydrochloride ligation.8,18 Open in a separate window Scheme 1 Protein Splicing (A), trans-Splicing (B) and EPL (C)ExN and ExC represent N- and C-exteins, respectively. IntN and IntC represent N- and C-intein fragments, respectively. Despite the many successes of EPL, the approach often suffers from low overall efficiency due to complications associated with the generation of protein -thioesters. In particular, fusions to inteins are, to varying extents, susceptible to premature extein cleavage, both and during initial purification from cell lysates, which reduces the isolated yield of the intein fusion Tamsulosin hydrochloride needed for the subsequent thiolysis step.19 Importantly, the cleaved extein side-product is unreactive toward EPL, and its separation from the desired -thioester or the ligation product is often difficult for large proteins, such as antibodies.19 To compound matters, the thiolysis reaction itself can be slow and inefficient, further strengthening the need to develop customized purification regimes, involving multiple chromatographic steps, to isolate the desired product from complex mixtures.20?22 Collectively, these technical issues mean that a considerable investment in time and resources is usually required before a semisynthetic protein is obtained in useful quantities. To overcome the various drawbacks associated with the intein thiolysis process central to EPL, we envisioned an alternative strategy based on naturally occurring split inteins. Unlike inteins used in standard EPL, which are contiguous polypeptides that catalyze protein splicing in (protein (Npu) split DnaE intein suitable for efficient -thioester generation. Specifically, we mutated the catalytic C-terminal residue in the IntC fragment (Asn137) and the first residue in the C-extein (Cys+1) to Ala, to allow for efficient build up of the desired splicing intermediates upon exposure to an N-extein-NpuN fusion (Physique ?(Figure1). Preliminary1). HES1 Preliminary studies showed that mixing N-extein-NpuN fusions (where N-extein corresponded to various model proteins) with the mutant NpuC (NpuCAA) led to highly efficient N-extein -thioester formation in a thiol-dependent manner (Figures S3 and S4). Importantly, only.