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

ABSTRACT: 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-His ₁₀ 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.

SUBMITTER: Hollinger J

PROVIDER: S-EPMC10245961 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Expression and purification of the mitochondrial transmembrane protein FAM210A in <i>Escherichia coli</i>.

Hollinger Jared J Wu Jiangbin J Awayda Kamel M KM O'Connell Mitchell R MR Yao Peng P

bioRxiv : the preprint server for biology 20230527

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-His <sub>10< ...[more]

PMID: 37292620

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Project description:E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.

Project description:BackgroundHepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential.ObjectivesThe aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate.Materials and methodsThe truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.ResultsThe subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media.ConclusionsIn this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

Dataset Information

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

Publications

Expression and purification of the mitochondrial transmembrane protein FAM210A in <i>Escherichia coli</i>.

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