N-terminal chemical protein labeling using the naturally split GOS-TerL intein.

Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site.

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int(N) fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int(N) fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int(C) fragment of the GOS-TerL intein.

Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing.

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

Chemical-tag labeling of proteins using fully recombinant split inteins.

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.

Generation of a genetically encoded, photoactivatable intein for the controlled production of cyclic peptides.

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-to-tail cyclized peptides and large libraries thereof (SICLOPPS=split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light-dependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, non-catalytic position is now reported. Stable intein precursors were purified from the E.coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.