Expression, purification and antibody preparation of recombinant human SM22alpha.
- Author:
Jin-kun WEN
1
;
Lei NIE
;
Jing-yi LI
;
Yun-hui CHENG
;
Mei HAN
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Animals; Genetic Vectors; Humans; Microfilament Proteins; biosynthesis; genetics; isolation & purification; Muscle Proteins; biosynthesis; genetics; isolation & purification; Pichia; metabolism; Plasmids; Rabbits; Rats; Recombinant Proteins; biosynthesis; genetics; isolation & purification
- From: Chinese Journal of Applied Physiology 2006;22(3):379-383
- CountryChina
- Language:Chinese
-
Abstract:
AIMThe recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.
METHODSUsing 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.
RESULTSThe 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.
CONCLUSIONHigh-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.
