Expression and purification of porcine Akirin2 in Escherichia coli
Akirin2 is a recently discovered gene related to immune responses that also plays an important role in skeletal myogenesis. In this study, in order to scale up the production of recombinant porcine Akirin2 (pAkirin2), we report the expression and purification of a His-tagged version of recombinant pAkirin2 in Escherichia coli. The pAkirin2 is a polypeptide of 203 amino acids containing 42 rare codons. The recombinant pAkirin2 was expressed as an N-terminal fusion protein with His6 tag in E. coli Rosetta (DE3) host strain. The protein was purified by Ni-IDA affinity chromatography, yielding over 90% highly purified recombinant pAkirin2, with a considerable yield of 4 mg/L. The purified recombinant pAkirin2 was confirmed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer analysis. The refolded purified recombinant pAkirin2 significantly promoted the proliferation of C2C12 cells, indicating that it was active. This report provides a reliable technique for recombinant expression and purification of pAkirin2 protein.
Expression and purification of porcine Akirin2 in Escherichia coli
Akirin2 is a recently discovered gene related to immune responses that also plays an important role in skeletal myogenesis. In this study, in order to scale up the production of recombinant porcine Akirin2 (pAkirin2), we report the expression and purification of a His-tagged version of recombinant pAkirin2 in Escherichia coli. The pAkirin2 is a polypeptide of 203 amino acids containing 42 rare codons. The recombinant pAkirin2 was expressed as an N-terminal fusion protein with His6 tag in E. coli Rosetta (DE3) host strain. The protein was purified by Ni-IDA affinity chromatography, yielding over 90% highly purified recombinant pAkirin2, with a considerable yield of 4 mg/L. The purified recombinant pAkirin2 was confirmed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer analysis. The refolded purified recombinant pAkirin2 significantly promoted the proliferation of C2C12 cells, indicating that it was active. This report provides a reliable technique for recombinant expression and purification of pAkirin2 protein.
___
- Beutler B, Moresco, EMY (2008). Akirins versus infection. Nat Immunol 9: 7–9. Buckingham M (2001). Skeletal muscle formation in vertebrates. Curr Opin Genet Dev 11: 440–448.
- Buckingham M (2006). Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr Opin Genet Dev 16: 525–532.
- Chen XL, Huang ZQ, Jia G, Wu XQ, Wu CM (2012). Molecular cloning, tissue distribution, and functional analysis of porcine Akirin Anim Biotechnol 23: 124–131.
- Chen XL, Huang ZQ, Wang H, Jia G, Liu GM, Guo XL, Tang RY, Long DB (2013). Role of Akirin in skeletal myogenesis. Int J Mol Sci 14: 3817–3823.
- Goto A, Matsushita K, Gesellchen V, EI Chamy L, Kuttenkeuler D, Takeuchi O, Hoffmann JA, Akira S, Boutros M, Reichhart JM (2008). Akirins are highly conserved nuclear proteins required for NF-κB-dependent gene expression in Drosophila and mice. Nat Immunol 9: 97–104.
- Grosjean H, Fiers W (1982). Preferential codon usage in prokaryotic genes: the optimal codon–anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18: 199–209.
- Kane JF (1995). Effects of rare codon clusters on high level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6: 494–500.
- Kim H, Lee SK, Hong MW, Park SR, Lee YS, Kim JW, Lee HK, Jeong DK, Song YH, Lee SJ (2013). Association of a single nucleotide polymorphism in the akirin2 gene with economically important traits in Korean native cattle. Anim Genet 44: 750–753.
- Macqueen DJ, Johnston IA (2009). Evolution of the multifaceted eukaryotic akirin gene family. BMC Evol Biol 9: 34.
- Macqueen DJ, Bower NI, Johnston IA (2010). Positioning the expanded akirin gene family of Atlantic salmon within the transcriptional networks of myogenesis. Biochem Biophys Res Commun 400: 599–605.
- Sasaki S, Yamada T, Sukegawa S, Miyake T, Fujita T, Morita M, Ohta T, Takahagi Y, Murakami H, Morimatsu F et al. (2009). Association of a single nucleotide polymorphism in akirin2 gene with marbling in Japanese Black beef cattle. BMC Res Notes 2: 131–135.
- Watanabe N, Satoh Y, Fujita T, Ohta T, Kose H, Muramatsu Y, Yamamoto T, Yamada T (2011). Distribution of allele frequencies at TTN g.231054C > T, RPL27A g.3109537C > T and AKIRIN2 c.*188G > A between Japanese black and four other cattle breeds with differing historical selection for marbling. BMC Res Notes 4: 10.
- Yin JC, Li GX, Ren XF, Herrler G (2007). Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 127: 335–347.