In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT
In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT
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DOI：
10.1046/j.1432-1327.2000.01073.x
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发表时间：
2000-02-01
期刊：
影响因子：
--
通讯作者：
Dekker, N
中科院分区：
文献类型：
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作者：
Kramer, RA;Zandwijken, D;Dekker, N
OmpT是存在于大肠杆菌外膜中的蛋白酶。在E.大肠杆菌中，使用T7系统，导致OmpT的积累作为包涵体。包涵体在尿素中溶解后，在洗涤剂N-十二烷基-N，N-二甲基-1-铵基-3-丙磺酸盐存在下，通过稀释可在体外折叠蛋白。脂多糖的加入是获得活性酶的必要条件。正确折叠的蛋白通过离子交换层析纯化至均一，总收率为57%。Lys 217-Arg 218之间的自身蛋白水解是纯化过程中的主要问题，但通过引入突变G216 K和K217 G可以消除降解。使用内部猝灭底物Abz-Ala-Arg-Arg-Ala-Tyr（NO2）-NH 2（其中Abz是邻氨基苯甲酰基，Tyr（NO2）是3-硝基酪氨酸）的新型荧光测定法能够测定动力学参数。野生型酶对底物的亲和力K-m为0.4 μ M，转换数k（cat）为40 s（-1）。双变异体的Km和k（cat）分别为1.1 μ M和1.6 s（-1）。野生型和变体的pH曲线是相同的，分别在pH 6.5和pK（α）值5.6和7.5下显示出最佳活性。圆二色光谱表明，这两种酶的β-链构象的含量高，并在此基础上提出了β-桶拓扑模型。
OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity K-m of 0.4 mu M for the substrate and a turnover number k(cat) of 40 s(-1). The K-m and k(cat) for the double variant were 1.1 mu M and 1.6 s(-1), respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pK(a) values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.