In order to better understand which amino acids are crucial for the bioconjugation mediated by the bTG enzyme, we produced eleven other mutants based on the CS_BSA2 identified conjugation sequence (CS_1 to CS_11, Figures S35 to S45 and Table ) and tested them for conjugation with the dansylcadaverine moiety (Figures S46 to S56, Table and Table ). Shortening the sequence to DAIPENL (CS_1) had no impact on the percentage of conversion. The four amino acids DAIP do not appear as essential for the recognition as a similar conjugation could be obtained with a random GSGS sequence (CS_6). The addition of three amino acids between the ENL-reactive site and the His-Tag led to full conversion (CS_5). On the other hand, when the size of the linker sequence between the reactive site and the monobody sequence was shortened, a diminution in conversion was observed (CS_2 and CS_3). Surprisingly, when the sequence was reduced to the "ENL" pattern, 100% conjugated product was obtained (CS_4). As a control, we also tested the "EQL" sequence, for which no conversion could be detected, therefore confirming the specific recognition of the asparagine over a glutamine (CS_7). Replacement of the glutamic acid (E) by an alanine led to a significant drop in conversion (CS_8) while its replacement by an aspartic acid (D) sharing similar reactivity and structure maintained moderate conversion (CS_11), albeit reduced compared with CS_4. The mutation of the leucine amino acid (L) by an alanine had less impact on the bioconjugation reaction (CS_9) whereas the mutation of both E and L by alanine had a deleterious effect on the formation of the conjugated product (CS_10). Taken together, these data suggest that while "ENL" might be sufficient to promote bioconjugation, local secondary or even secondary structural elements also play a role.