Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Abstract

<jats:title>Abstract</jats:title><jats:p>Manipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, Na<jats:sub>V</jats:sub>1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.</jats:p>

The current chemical biology tool box for studying ion channels

“…Furthermore, both approaches rely on ribosomal tolerance, which means certain backbone or side chain types cannot be incorporated (see section on Semi-synthetic approaches below). Lastly, and depending on the sequence context, incorporation efficiency can be highly variable, unwanted re-initiation from non-canonical start codons can occur or introduced stop codons can be read through, resulting in non-specific incorporation of (endogenous) amino acids at the site of interest (Kalstrup & Blunck, 2015;Lynagh et al 2018Lynagh et al , 2020Poulsen et al 2019;Khoo et al 2020). The latter issues are particularly problematic in the most N-and C-terminal parts of proteins.…”

“…The three components assemble (centre panel) and the split inteins excise themselves, yielding a full-length ion channel carrying the modifications inserted into the synthetic peptide (right panel). B, cartoon of a synthetic peptide (centre) including chemical structures of the post-translational modifications used by Khoo et al 2020: a Lys side chain replaced by a thio-acetylated Lys derivative (tAcLys, mimicking an acetylated Lys, but with increased metabolic stability; left panel) and a Tyr replaced by a phosphonylated Tyr analogue (phTyr, which cannot be hydrolyzed; right panel). mechanism of C-type inactivation in semisynthetic KcsA and archaebacterial K v (K V AP) channels (Devaraneni et al 2013;Matulef et al 2013) and to incorporate isotope labels to perform 2D infrared spectroscopy on semi-synthetic KcsA channels (Kratochvil et al 2016).…”

“…We have recently used this approach to study P2X receptors carrying non-canonical lysine derivatives in the ATP binding pocket, as well as Na V channels carrying single post-translational modifications (phosphorylations, thioacetylations, methylations) or combinations thereof in the intracellular domain linkers (Fig. 5B) (Khoo et al 2020). This represented the first insertion of two metabolically stable post-translational modification mimics, thus paving the way to assess their effects on ion channel function and pharmacology, including a possible interplay of the modifications.…”

“…For example, insertion of synthetic transmembrane segments is likely to be challenging, as is implementation in sequence contexts that generally lead to low yields (e.g. tandem protein trans-splicing in extracellular domains) (Khoo et al 2020). Yet, the ever-decreasing costs associated with gene and peptide synthesis mean that these technologies are likely to become more prevalent in the future, as this trend obviates the need for specialized equipment, e.g.…”

“…tandem protein trans‐splicing in extracellular domains) (Khoo et al . 2020). Yet, the ever‐decreasing costs associated with gene and peptide synthesis mean that these technologies are likely to become more prevalent in the future, as this trend obviates the need for specialized equipment, e.g.…”