Pan Hai-Yun, Cheng Ying, Zhu Su-Wen, Fan Jun
School of Life Sciences, Anhui Agricultural University, Hefei , 230036, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Mar;23(2):206-10. doi: 10.1016/s1872-2075(07)60016-8.
S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase (SUMT) is a novel red fluorescence indicator. However, the production of SUMT in Escherichia coli is restricted by its relatively low solubility, and little is known about the red fluorescent materials that are associate with SUMT. Two individual SUMT mutations, L166A and L88R/L89G double mutant were produced by site-directed mutagenesis. Both mutants were overexpressed in E. coli and purified by Ni-NTA chromatography. The reddish mixtures isolated from the purified L88R/L89G double mutant were analyzed by UV-visible spectra scanning and mass analysis(MS). The L88R/L89G double mutant has enzymatic activity in vivo, whereas L166A mutant loses the activity. Trimethylpyrrocorphin is identified as the main constituent in the isolated pigments. The purified L88R/L89G mutant increases protein solubility, which is applied potentially as the fluorescent indicator denoting the solubility of protein fusion partner.
S-腺苷甲硫氨酸依赖性尿卟啉原III甲基转移酶（SUMT）是一种新型红色荧光指示剂。然而，SUMT在大肠杆菌中的产生受到其相对较低溶解度的限制，并且关于与SUMT相关的红色荧光物质知之甚少。通过定点诱变产生了两个单独的SUMT突变体，即L166A和L88R/L89G双突变体。这两个突变体均在大肠杆菌中过表达，并通过镍-亚氨基二乙酸（Ni-NTA）色谱法进行纯化。通过紫外-可见光谱扫描和质谱分析（MS）对从纯化的L88R/L89G双突变体中分离出的微红混合物进行了分析。L88R/L89G双突变体在体内具有酶活性，而L166A突变体则失去了活性。三甲基卟吩被鉴定为分离出的色素中的主要成分。纯化的L88R/L89G突变体增加了蛋白质的溶解度，其有可能用作表示蛋白质融合伴侣溶解度的荧光指示剂。
