Fraser P D, Linden H, Sandmann G
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Biochem J. 1993 May 1;291 ( Pt 3)(Pt 3):687-92. doi: 10.1042/bj2910687.
The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.
聚球藻八氢番茄红素去饱和酶已从大肠杆菌过表达菌株中分离出来。血浆pPDSde135直接介导全长多肽的过表达。重组蛋白占总细胞蛋白的5%，主要存在于包涵体部分。尿素用于从包涵体部分溶解重组蛋白，随后该蛋白在DEAE-纤维素柱上纯化至同质。纯化方案在20倍纯化后产生4.0mg同质去饱和酶蛋白，从100ml大肠杆菌悬浮培养物中回收了40%的原始蛋白。重组去饱和酶在SDS/PAGE上的表观分子量为53kDa，并与针对表达蛋白产生的抗血清发生交叉反应。去除尿素后去饱和酶活性得以恢复。该酶催化八氢番茄红素通过六氢番茄红素转化为ζ-胡萝卜素。去饱和酶反应的这些产物主要以顺式构型存在。脂质补充增强了活性。观察到NAD+和NADP+参与其中，而FAD是无效的电子受体。
