Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli

• Published in: Protein expression and purification Vol. 210; p. 106322
• Main Authors: Hollinger, Jared, Wu, Jiangbin, Awayda, Kamel M., O'Connell, Mitchell R., Yao, Peng
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2023
• Subjects: Escherichia coli - genetics; Escherichia coli - metabolism; FAM210A; HEK293 Cells; Humans; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mitochondria; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Peptide Elongation Factor Tu - chemistry; Peptide Elongation Factor Tu - genetics; Peptide Elongation Factor Tu - metabolism; Protein synthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Translation; Transmembrane; FAM210A; Mitochondria; Translation; Protein synthesis; Transmembrane
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2023.106322

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10 fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein. •First expression and purification of mitochondrial transmembrane protein FAM210A.•Develop a method to purify human FAM210A without mitochondrial targeting sequence.•Validate the functionality of purified FAM210A interacting with human EF-Tu.•FAM210A forms complexes with E.coli derived EF-Tu during overexpression.