We further describe two-site modification of an IgG1 protein in a one-pot reaction

We further describe two-site modification of an IgG1 protein in a one-pot reaction. of an unnatural amino acid into the protein is possible, but this requires manipulation of the genetic code, involves substantial optimization and therefore adjustments on a case by case basis.47A further alternative consists of complete synthesis of the polypeptide of interest (POI) by chemical means, or the combination of various segments through chemical ligation. Although this approach enables site-specific incorporation of the desired modifications, such technologies are limited by the size of the POI: target POIs are usually limited to 250 AA or less.812 We and others have used a peptide ligase, sortase A, for a variety of protein modifications1318Sortase A recognizes a short amino acid sequence, LPXTGG, where X is any amino acid except proline. To this sequence, Sortase A will ligate a peptide that contains an N-terminal oligo-glycine stretch, equipped at its C-terminus with any cargo of choice. The LPXTGG recognition motif is easily incorporated CMPDA into a POI (Scheme 1A) and is generally placed at or near the C-terminus. Sortase-mediated labeling is robust and finds use in a broad range of applications. Tam and CMPDA coworkers discovered another ligase, butelase-1, in the seed pods of the plantClitoria ternatea, purified it, and used it for the modification and cyclization of peptides as well as proteins.1923Compared to sortase-A, butelase-1 obeys a distinct set of recognition rules. Butelase-1 also has a much higher turnover number. Butelase 1 recognizes a AsnHisVal (NHV) motif, which it ligates to incoming nucleophiles composed of two amino acids to which a cargo of choice may be attached, provided two rules are followed. The N-terminal residue can be any one except proline and the n-1 terminal residue must be a Cys, Ile, Leu or Val (Scheme 1B). == Scheme 1. == Sortase A and Butelase 1 ligation mechanisms and our work described in this paper. The distinct rules obeyed by these ligases allows a combination of sortase A and butelase 1 in a single reaction to prepare unnatural C-to-C fusions of two different proteins and to label proteins at two distinct sites in a one-pot reaction (Scheme 1C). VHHs, also called nanobodies, are small proteins (~14 kDa), that correspond to the antigen binding domain of heavy chain-only ENAH camelid antibodies. They are the smallest antibody fragments that retain antigen recognition. We used two such VHHs: VHH7, which recognizes class II MHC products (MHC II) and VHH-Enh, which recognizes eGFP. CMPDA Generally, the production of bispecific antibodies or their derivatives is a desirable goal from the perspective of therapeutic applications. In this paper, we chose two VHHs of different specificity as a proof-of-concept. These VHHs were modified to carry the desired motifs: VHH7 bears the LPETGG motif at the C-terminus, while VHH-Enh has the NHV motif at the C-terminus. We synthesized a two-headed, PEG-based linker compatible with both sortase A and butelase-1 by standard SPPS (Figure 1A). VHH7 (75 M) was incubated at 4 C with the linker (500 M) and sortase A (2 M) for 15 hours. Analysis by mass spectrometry of the reaction mixture showed no remaining His-tagged VHH7, indicating full conversion for the first ligation. The His-tagged sortase was removed by depletion on Ni-NTA agarose beads and remaining free linker was removed from the ligation product through centrifugation-based size exclusion or alternatively by dialysis. To the ligated product (250 M) VHH-Enh (25 M) and butelase 1 (1.0 M) were added and incubated at 37 C for 6 hours with agitation. The excess of starting material is necessary in order to reach complete conversion. The reaction mixture was purified either by FPLC or by centrifugation-based size exclusion to afford.