Li D H, Xi H, Yu X B, Cai Y P
College of Life Sciences, Anhui Agricultural University, Hefei, China.
Genet Mol Res. 2015 Dec 9;14(4):16535-45. doi: 10.4238/2015.December.9.25.
The Arabidopsis thaliana genome encodes 56 subtilisin-like serine proteases (subtilases). In order to evaluate the protease activity of a previously uncharacterized subtilase, designated as AtSBT1.9, we cloned its full-length cDNA from A. thaliana seedlings. An AtSBT1.9 mature peptide coding sequence was inserted into the bacterial expression vector, pMAL-c2x, and the recombinant vector was transformed into Escherichia coli BL21 (DE3). The recombinant AtSBT1.9 tagged by maltose binding protein (MBP) was induced as a 117.5-kDa protein in the soluble form in E. coli BL21 (DE3). MBP-AtSBT1.9 was expressed at a level of 11% (w/w) of the bacterial total protein. Protein purification using Amylose Resin revealed a recombinant AtSBT1.9 protease activity of 9.23 U/mg protein at pH 7 and 25°C. Maximal activity occurred over a broad pH (7-8) and temperature (25°-42°C) optimal range. Validation of AtSBT1.9 protease activity would help in characterizing its in vivo function in A. thaliana.
拟南芥基因组编码56种枯草杆菌蛋白酶样丝氨酸蛋白酶（枯草杆菌蛋白酶）。为了评估一种先前未鉴定的枯草杆菌蛋白酶（命名为AtSBT1.9）的蛋白酶活性，我们从拟南芥幼苗中克隆了其全长cDNA。将AtSBT1.9成熟肽编码序列插入细菌表达载体pMAL-c2x中，并将重组载体转化到大肠杆菌BL21(DE3)中。在大肠杆菌BL21(DE3)中，由麦芽糖结合蛋白（MBP）标记的重组AtSBT1.9以可溶性形式被诱导表达为一种117.5 kDa的蛋白质。MBP-AtSBT1.9的表达量占细菌总蛋白的11%（w/w）。使用直链淀粉树脂进行蛋白质纯化显示，在pH 7和25°C条件下，重组AtSBT1.9的蛋白酶活性为9.23 U/mg蛋白质。最大活性出现在较宽的pH（7 - 8）和温度（25° - 42°C）最佳范围内。验证AtSBT1.9的蛋白酶活性将有助于表征其在拟南芥中的体内功能。
