Expression, purification and antibody preparation of recombinant human SM22alpha / 中国应用生理学杂志

ABSTRACT

<p><b>AIM</b>The recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.</p><p><b>METHODS</b>Using pGEM3z-SM22alpha as the template, SM22alpha coding region was amplified by PCR, and was inserted the expression vector pPIC9. Then the recombinant plasmid pPIC9-SM22alpha was transfected into Pichia pastoris. The products induced by methanol were precipitated by ammonium sulfate, then CM-cellulose chromatography was performed for SM22alpha. Polyclonal antibody against SM22alpha was produced by immunizing a rabbit with purified recombinant SM22alpha.</p><p><b>RESULTS</b>The positive clone with SM22alpha got high output at 84 hours after induction by methanol. The SM22alpha prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. Polyclonal antibody against SM22alpha could detect the SM22alpha expression in human or rat vascular walls.</p><p><b>CONCLUSION</b>High-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.</p>