In order to investigate the potential of the bTG transglutaminase to conjugate a primary amine in a sequence specific manner, we chose the monobody Mb(Lck_3) as a model target. This antibody mimetic is able to bind to the lymphocyte specific protein tyrosine kinase domain 2 (Lck-SH2 domain), an oncoprotein playing an important role in several cancers. The monobody was expressed in E. coli first with a C-terminal TEV-HIS tag (Mb(Lck_3)_CHis, Figure and Table ). The bioconjugation reaction was tested with a dansylcadaverine as an amine donor, which enables easy detection at 335 nm and a home-made bTG purified enzyme (Figure and Table ). The bioconjugation reaction led to no formation of the desired product (expected mass +318, Figure and Table ). We then produced Mb(Lck_3)_CHis_bTG (Figure and Table ), which includes the reported recognition sequence YAHQAHY between the monobody sequence and the TEV site. The bioconjugation reaction with this new sequence led to the desired product with an estimated 65% conversion (Figure , Table and Table ). To confirm that the conjugation site was located on the glutamine of the reported recognition sequence, a Q100A mutant was prepared (Figure and Table ), in which the glutamine was replaced by an alanine. Surprisingly, a conjugated product could be detected and in higher yields compared to the model Mb(Lck_3)_CHis_bTG (Figure , Table and Table ), indicating that the bioconjugation could be driven by other factors. The difference in reactivity between Mb(Lck_3)CHis and Mb(Lck_3)_Q100A could be due to the presence of the YAHAAHY linker, which could provide a change in the monobody structure and grant better accessibility to the reactive site. This result led us to investigate other sequences to better understand the selectivity of the bTG enzyme and Mb(Lck_3)_NHis, possessing a His-Tag and a TEV site at the N-terminus (Figure and Table ) was tested. Surprisingly, even in the absence of the recognition sequence, the conjugated product was formed at 57% (Figure , Table and Table ). Replacement of the glutamine amino acid contained in the TEV site by an alanine (mutant Q13A, Figure and Table ) also led to conversion (Figure , Table and Table ). As asparagine and glutamine share similar reactivity and structure, we also created the N9A mutant (Figure and Table ) where the asparagine (N) was replaced by an alanine (A). With this sequence, no product formation could be observed (Figure , Table and Table ) and the same result was found when both the asparagine and the glutamine were mutated by an alanine amino acid (N9A_Q13A, Figures S14, S15, Tables and).