共转化大肠杆菌hemA和hemL基因增强小麦过氧化物酶WP1在原核系统中的功能性表达

Enhancement of functional expression of wheat peroxidase WP1 in prokaryotic system by co-transforming with hemA and hemL of Esherichia coli

摘要:

小麦种子过氧化物酶WP1属于含血红素的植物Ⅲ型过氧化物酶，该酶不仅具有抗真菌活性，而且影响面粉加工品质。为提高WP1在大肠杆菌中的功能性表达，构建了用于提高大肠杆菌内源血红素合成，包含hemA和hemL基因的重组质粒pACYC-A-L，将其分别与包含WP1基因的分泌型和非分泌型表达载体pMAL-p4x-WP1与pET21a-MBP-WP1共同转化大肠杆菌T7 Express菌株；利用直链淀粉 (Amylose) 亲和层析柱纯化获得MBP-WP1融合蛋白，并以2,2’-联氮-二 (3-乙基苯并噻唑-6-磺酸

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.