A mesophilic cysteine-less split intein for protein trans -splicing applications under oxidizing conditions

Abstract

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.

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

“…In fact, many groups have repeatedly observed yields of 10% or less with ncAA incorporation into transporters 36 , ion channels [37][38][39] , and G protein-coupled receptors 40,41 . Although the generally low yields observed with tPTS likely restrict the approach to applications that do not require large amounts of protein, at least some of the above limitations can be addressed by engineering more promiscuous and efficient split inteins [10][11][12]42 or by adding affinity tags to promote split intein interactions 18 . Such improvements would allow the approach to be applied to a broader complement of target proteins.…”

Intein mediated high throughput screening for bispecific antibodies

“…These split inteins, like the recently engineered cysteine-less split intein Aes, might be an alternative for generating reconstituted antibodies for sensitive cellular assays not compatible with the reactants TCEP and DHAA as, for instance, applied in effector cell recruitment screenings. [45] TCEP and DHAA do not cause a luminescence signal detection in the CD40 activation assay, but reconstituted antibody variants require the addition of a non-native six amino acid sequence within the hinge for PTS activation, which might alter the geometry and flexibility of the molecule compared to the natural IgG reference molecule. The additional included cysteine might lead to potential new HC -HC or HC -LC disulfide bonds after reduction and reoxidation procedures, as a result of forming a byproduct, and might account for lower maximal extent of CD40 activation compared to the reference antibody.…”

The current chemical biology tool box for studying ion channels

“…This capability has been harnessed to assemble partial, non-functional Ca V and Na V channel fragments into full-length, functional channels using a single pair of split inteins (Subramanyam et al 2013;Lueck et al 2016). Given the increasing number of identified and splicing-optimized split inteins (Stevens et al 2016(Stevens et al , 2017Gramespacher et al 2018;Bhagawati et al 2019;Burton et al 2020;Pinto et al 2020), it is now also possible to use two orthogonal pairs of split inteins to insert a synthetic peptide between recombinantly expressed N-and C-terminal protein fragments (Fig. 5A).…”