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

Experimental & Molecular Medicine 2021년 53권 1호 p.115 ~ 124
김효준, 이진행, 이기백, Shin Ji-Woong, 권미애, 이수진, Jeong Eui-Man, 조성엽, 김인규,
소속 상세정보
김효준 ( Kim Hyo-Jun ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
이진행 ( Lee Jin-Haeng ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
이기백 ( Lee Ki-Baek ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
 ( Shin Ji-Woong ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
권미애 ( Kwon Mee-Ae ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
이수진 ( Lee Soo-Jin ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
 ( Jeong Eui-Man ) - Jeju National University College of Pharmacy Department of Pharmacy
조성엽 ( Cho Sung-Yup ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology
김인규 ( Kim In-Gyu ) - Seoul National University College of Medicine Department of Biochemistry and Molecular Biology

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 tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein?protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.

키워드

Expression systems; Protein purification

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