With these promising results, we expressed other Mb(Lck_3) mutants in order to identify the minimal sequence needed. As the bioreaction with an ENL-containing motif (TEV sequence) straight after the monobody sequence did not lead to any conversion (Mb(Lck_3)_CHis), whereas conversion could be observed when the recognition sequence was inserted in between (Mb(Lck_3)_bTG_CHis or Q100A mutant), we hypothesized that the structural accessibility of the conjugation site was an important factor. Therefore, we designed our new mutant sequences with a small, five amino-acid linker (LPETG) between the monobody sequence, which was available from previous investigations (data not shown) and the insert sequence. We then produced four mutants with the peptide sequences previously tested (CS_BSA1, CS_BSA2, CS_Control1, CS_Control2, Figures S27 to S30 and Table ). Bioconjugation reactions followed the same trend as for the peptide sequences alone, with conjugated product formed only in the presence of the ENL-containing sequence (DAIPENLPPLTADFAEDK) and similar conversion (94% conversion, Figures S31 to S34, Table and Table ).