Precision crosslinking and active-site modifications via site-specific protein engineering

First, a site-specific method for covalently and site-specifically attaching two proteins under biocompatible conditions was developed. The method combined the sortase-mediated installation of a peptide containing a cysteine-reactive functionality onto the N-terminus of an antigen binding protein followed by a subsequent chemoselective conjugation. Investigation of the promiscuity of sortase, a cysteine-dependent transpeptidase, for substrates containing cysteine-alkylating reagents suggested good tolerance of the sortase for such electrophiles. Multiple peptides each containing a sortase recognition sequence and a different cysteine-selective electrophile were therefore synthesized via solid-phase peptide synthesis (SPPS). Significant optimization of the sortase reaction was required to yield sufficient product for functional assays. Speeding up the sortase reaction using kinetically enhanced mutants was found to enhance the production of product material, likely by counteracting the concurrent alkylation of the sortase active site by the electrophilic peptide substrate. After successfully attaching cysteine-targeting moieties onto several antigen-binding protein, so-called “nanobodies” using sortase transpeptidation, site-specific conjugation to cysteine-containing proteins was optimized to generate useful yields of products with new functionalities. Conjugation was shown to be unsuccessful without prior purification of the sortase reaction, likely due to the presence of competing crosslinking partners and loss of the transpeptidation product over time in the presence of the sortase. Sortase-mediated electrophile installations were performed on nanobodies (with and without endogenous cysteines) and crosslinked to other nanobodies, GFP, and even intact IgG antibodies to yield bi-specific or mono-specific, fluorescent constructs combining the properties of multiple intact proteins. Final conjugate yields were low after multiple purification steps. The function of both protein entities within all final constructs were confirmed using fluorescence microscopy. In the second part, amber codon suppression was combined with the chemoselective Staudinger-phosphite reaction to enable the selective caging of nanobody active sites with a light-cleavable PEG group. Decaging was envisioned to progress via UV-induced photolysis of an ortho-nitrobenzyl polyethylene glycol (ONB-PEG) functionality, followed by hydrolysis to yield a final aniline. An azido-tyrosine was introduced at the active site of a GFP binding nanobody using amber suppression. To test the activity of the decaged product, TCEP reduction successfully converted the incorporated azide to the corresponding aniline, which was then subjected to a substrate binding assay. Unfortunately, the binding affinity of the decaged nanobody was only partially restored to that of the wild-type nanobody. The azide was subsequently functionalized with a light-cleavable ONB-PEG-phosphite via the Staudinger-phosphite reaction in aqueous buffer. According to a recent literature survey, this represents the first example of a Staudinger-phosphite reaction performed on a nanobody under biological conditions. Caging was shown to prevent GFP-nanobody interaction via a GFP binding assay utilizing the fluorescence-enhancing properties of the chosen nanobody. Cleavage of the ONB group was achieved using UV irradiation, and the resulting decaged construct was shown to possess partially restored GFP binding affinity.