Dekker N, Merck K, Tommassen J, Verheij H M
Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
Eur J Biochem. 1995 Aug 15;232(1):214-9. doi: 10.1111/j.1432-1033.1995.tb20801.x.
Recombinant outer-membrane phospholipase A (OMPLA) of Escherichia coli was expressed without its signal sequence from the T7 phi 10 promoter. As a result of the cloning strategy the protein had an N-terminal extension of six amino acid residues. The protein accumulated in the cytosol in inclusion bodies. Conditions were established for the efficient folding of OMPLA in vitro in the presence of Triton X-100. After in vitro folding, the protein was present as a mixture of folded and unfolded forms. Ion-exchange chromatography was used for the purification of OMPLA and the separation of correctly folded, enzymically active enzyme from unfolded inactive protein. The final protein preparation was pure and fully heat-modifiable based on SDS/PAGE. The recombinant enzyme had a specific activity of 71 U/mg, which is similar to the value of the wild-type enzyme, purified from the membrane. The final yield of active enzyme was 35 mg protein/l culture of an A600 of 6. Circular dichroism spectroscopy revealed a high content of beta strand, in good agreement with a predicted beta-barrel structure of this outer-membrane protein.
大肠杆菌重组外膜磷脂酶A（OMPLA）在无信号序列的情况下由T7噬菌体10启动子表达。由于克隆策略，该蛋白有一个六个氨基酸残基的N端延伸。该蛋白以包涵体形式积聚在细胞质中。建立了在Triton X-100存在下OMPLA体外高效折叠的条件。体外折叠后，该蛋白以折叠和未折叠形式的混合物存在。离子交换色谱用于OMPLA的纯化以及从未折叠的无活性蛋白中分离正确折叠的、具有酶活性的酶。基于SDS/PAGE，最终的蛋白制品是纯的且完全可热修饰的。重组酶的比活性为71 U/mg，与从膜中纯化的野生型酶的值相似。活性酶的最终产量为每升A600为6的培养物35 mg蛋白。圆二色光谱显示β链含量高，与该外膜蛋白预测的β桶结构高度一致。
