Prokaryotic expression, purification, 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 the specification of myofiber 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 efficient expression with 2 mM IPTG for 2 h at 30 °C, yielding approximately 4.6 mg/L. The protein was then purified and identified by western blot, and it was used for preparing its polyclonal antibody. The recombinant pSix1 was tagged with a 6-His tag at its C-terminus, which could be conveniently purified by affinity column. The purified recombinant protein was used for immunizing Sprague Dawley rats to obtain the polyclonal antibody against pSix1. The antibody titer and specificity were determined by ELISA and western blot analysis, respectively. The tissue distribution of pSix1 was determined by western blot using the prepared polyclonal antibody.