Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody–epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.
The orthosteric ATP-binding site of the P2X receptors
is poorly
understood. Only a few compounds were well characterized for their
P2X receptor functional activity and subtype selectivity. This study
represents the first fully functional characterization of various
ATP derivatives combined with in silico studies to advance the understanding
of SARs at the orthosteric binding sites of P2X receptors leading
to the identification of 2-chloro-3-trifluoromethylbenzoyl ATP ester
as a novel pan-P2X receptor agonist and several subtype-selective
P2X receptor agonists. Furthermore, esterification of both hydroxyl
functions of ATP using 1-naphthoic acid has led to compound 26 acting as an antagonist at P2X1-4 and P2X2/3 receptors
and an agonist at P2X7 receptors. This particular ATP derivative will
allow interrogating the P2X7 receptor function while antagonizing
all other P2X receptor subtypes and therefore serve as a valuable
pharmacological tool in the future.
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