Protein size differentiation in ADAMTS7 Turbo ID experiment

Abstract

Background: In research for ADAMTS7’s substrate, a question arose about the size of Turbo ID constructs. Turbo ID is an enzyme that can catalyze the biotinylation of endogenous protein, which fuses to the protein of interest to cause an interaction with the proteins or a protein complex. Biotinylation is the process of attaching biotin to protein. This technique involves enzyme catalyzed Proximity Labeling (PL). PL assay is performed by labeling the protein of interest with an enzyme which biotinylates biomolecules in a proximity dependent manner. Turbo ID’s technology serves as a convenient protein - protein interaction detection method. Differences in sizes of the protein should be observed based on variations of ADAMTS7 being added to the biotinylation protein.

Method: After confirming the plasmid sequences were correct by DNA sequencing, the plasmids Del Pigeon, E151, E151Q ,E389 and E389Q were transfected into HEK 293 cells. The transfected cells were placed in Freestyle media for 48 hours for expression. The conditioned media was then harvested and purified using Q-Sepharose. The conditioned, purified media was run on a Western blot and imaged for ADAMTS7 protein bands.

Result: Del pigeon, E151, E151Q, E389Q and E389 has 256kDa, 290kDa, 290kDa, 307kDa and 307kDa respectively. The ones with higher kilo-Dalton has a bigger size compared to the one with the lesser kiloDalton.

Conclusion: We anticipate the Del pigeon with 256kDa will migrate faster towards the anode while E151, E151Q , E389Q and E389 will migrate more slowly because of their bigger sizes.