Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein-protein interactions of the fused protein.

ABSTRACT: Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein-protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST ta

SUBMITTER: Kim HJ 

PROVIDER: S-EPMC8080825 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature