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

Abstract

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.