Fig. 7
From: Retro-protein XXA is a remarkable solubilizing fusion tag for inclusion bodies
Functional verification of fusion proteins XXA-NbALFA and XXA-bdNEDP1. a The purified bdNEDP1 enzyme that fuses expression with the XXA tag was used to digest the substrate to test whether bdNEDP1 was active. The amount of substrate was fixed at 100Ā Ī¼mol, with lanes 1 and 7 being the control group without the enzyme. Ten Ī¼mol bdNEDP1 was used in the reactions of lanes 2 and 8, 1Ā Ī¼mol bdNEDP1 was used in the reactions of lanes 3 and 9, 100Ā nmol bdNEDP1 was used in the reactions of lanes 4 and 10, 10Ā nmol bdNEDP1 was used in the reactions of lanes 5 and 11, and 1Ā nmol bdNEDP1 was used in the reactions of lanes 6 and 12. The digestion reactions in lanes 1 to 6 was performed at 4Ā Ā°C, while the reactions in lanes 7 to 12 were performed at 25Ā Ā°C. The reaction time was fixed at 1Ā h. Whether bdNEDP1 was functional was judged by the reduction of the substrate and the increase in products. b The purified NbALFA nanobody that fuses expression with the XXA tag was used as the primary antibody to recognize its antigen to test whether NbALFA was functional. The substrate of the experimental group was Aspergillus flavus expressing the ALFA-tagged protein, while the control group was Aspergillus flavus not expressing the ALFA-tagged protein (empty vector control). The presence of a positive band that only exists in the experimental group indicates specificity, verifying that NbALFA was functional