Wanxue WEN, Shuai CHANG, Xiaoyan WANG, Junqiu LUO, Meng XU, Xiaoling CHEN, Zhiqing HUANG, Guangmang LIU, Daiwen CHEN, Bing YU

Prokaryotic expression, purifcation, polyclonal antibody preparation, and tissue distribution of porcine Six1

Sine oculis homeobox 1 (Six1), a member of the Six homeoproteins, plays an important role in skeletal myogenesis and thespecifcation of myofber diversity. In this study, in order to scale up the production of recombinant porcine Six1 (pSix1), a pET-30a(+)-pSix1 plasmid was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant pSix1 could be induced for efcientexpression with 2 mM IPTG for 2 h at 30 °C, yielding approximately 4.6 mg/L. The protein was then purifed and identifed by westernblot, and it was used for preparing its polyclonal antibody. The recombinant pSix1 was tagged with a 6-His tag at its C-terminus, whichcould be conveniently purifed by afnity column. The purifed recombinant protein was used for immunizing Sprague Dawley rats toobtain the polyclonal antibody against pSix1. The antibody titer and specifcity were determined by ELISA and western blot analysis,respectively. The tissue distribution of pSix1 was determined by western blot using the prepared polyclonal antibody.

PDF

___

Bessarab DA, Chong SW, Srinivas BP, Korzh V (2008). Six1a is required for the onset of fast muscle diferentiation in zebrafsh. Dev Biol 323: 216228.
Chen XL, Huang ZQ, Zhou B, Wang H, Jia G, Qiao JY (2014). Expression and purifcation of porcine Akirin2 in Escherichia coli. Turk J Biol 38: 339345.
Ford HL, Landesman-Bollag E, Dacwag CS, Stukenberg PT, Pardee AB, Seldin DC (2000). Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein. J Biol Chem 275: 2224522254.
Grifone R, Laclef C, Spitz F, Lopez S, Demignon J, Guidotti JE, Kawakami K, Xu PX, Kelly R, Petrof BJ et al. (2004). Six1 and Eya1 expression can reprogram adult muscle from the slow- twitch phenotype into the fast-twitch phenotype. Mol Cell Biol 24: 62536267.
Henckel P, Oksbjerg N, Erlandsen E, Barton-Gade P, Bejerholm C (1997). Histo- and biochemical characteristics of the Longissimus dorsi muscle in pigs and their relationships to performance and meat quality. Meat Sci 47: 311321.
Hetzler KL, Collins BC, Shanely RA, Sue H, Kostek MC (2014). Te homoeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle. Acta Physiol 210: 415 428.
Laclef C, Hamard G, Demignon J, Souil E, Houbron C, Maire P (2003). Altered myogenesis in Six1-defcient mice. Development 130: 22392252.
Liu Y, Li M, Ma J, Zhang J, Zhou C, Wang T, Gao X, Li X (2013). Identifcation of diferences in microRNA transcriptomes between porcine oxidative and glycolytic skeletal muscles. BMC Mol Biol 14: 7.
Maltin CA, Warkup CC, Matthews KR, Grant CM, Porter AD, Delday MI (1997). Pig muscle fbre characteristics as a source of variation in eating quality. Meat Sci 47: 237248.
Niro C, Demignon J, Vincent S, Liu Y, Giordani J, Sgarioto N, Favier M, Guillet-Deniau I, Blais A, Maire P (2010). Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome. Dev Biol 338: 168182.
OConnor PM, Bush JA, Suryawan A, Nguyen HV, Davis TA (2003). Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab 284: E110E119.
Schiafno S, Reggiani C (2011). Fiber types in mammalian skeletal muscles. Physiol Rev 91: 14471531.
Wang H, Chen XL, Huang ZQ, Zhou B, Jia G, Liu G, Zhao H (2014). Expression and purifcation of porcine PID1 gene in Escherichia coli. Turk J Biol 38: 523527.
Wang L, Chen X, Zheng Y, Li F, Lu Z, Chen C, Liu J, Wang Y, Peng Y, Shen Z et al. (2012). MiR-23a inhibits myogenic diferentiation through down regulation of fast myosin heavy chain isoforms. Exp Cell Res 318: 23242334.
Wu C, Wang Y, Zou M, Shan Y, Yao G, Wei P, Chen G, Wang J, Xu D (2007). Prokaryotic expression, purifcation, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14. Protein Expr Purif 56: 17.
Wu W, Ren Z, Chen C, Liu Y, Zhang L, Chao Z, Zuo B, Xu D, Lei M, Xiong Y (2012). Subcellular localization of diferent regions of porcine Six1 gene and its expression analysis in C2C12 myoblasts. Mol Biol Rep 39: 999510002.
Wu W, Ren Z, Liu H, Wang L, Huang R, Chen J, Zhang L, Li P, Xiong Y (2013). Core promoter analysis of porcine Six1 gene and its regulation of the promoter activity by CpG methylation. Gene 529: 238244.
Wu W, Ren Z, Wang Y, Chao Z, Xu D, Xiong Y (2011). Molecular characterization, expression patterns and polymorphism analysis of porcine Six1 gene. Mol Biol Rep 38: 26192632.
Xu PX, Zheng W, Huang L, Maire P, Laclef C, Silvius D (2003). Six1 is required for the early organogenesis of mammalian kidney. Development 130: 30853094.
Zheng HL, Li H, Sun YS, Yang ZY, Yu Q (2014). Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purifcation, and preparation of a polyclonal antibody. Genet Mol Res 13: 64486454.