title: [Enhancement of functional expression of wheat peroxidase WP1 in prokaryotic system by co-transforming with hemA and hemL of Esherichia coli].
abstract: Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Liwei
authors.initials: L
authors.lastname: Shan
authors.forename: Shuaikun
authors.initials: S
authors.lastname: Su
authors.forename: Yanni
authors.initials: Y
authors.lastname: Nan
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Guo
authors.forename: Sanhong
authors.initials: S
authors.lastname: Fan
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006418:Heme / Q000096:biosynthesis / Q000235:genetics*; D010544:Peroxidases / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D011993:Recombinant Fusion Proteins; D006418:Heme; D010544:Peroxidases; C070865:wheat peroxidase