Ghag S K, Brems D N, Hassell T C, Yeh W K
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Biotechnol Appl Biochem. 1996 Oct;24(2):109-19.
The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.
顶头孢霉双功能脱乙酰氧基头孢菌素C合成酶/羟化酶基因被克隆，并在重组大肠杆菌颗粒中作为不溶性无活性酶过度表达。只需一个凭经验优化的复性步骤，就能直接从颗粒提取物中回收约40%-60%预期的合成酶活性以及50%-80%的蛋白质纯度。在含有变性浓度尿素的情况下，通过单一阴离子交换色谱步骤进一步纯化至同质。将同质的未折叠蛋白转化为活性酶的主要障碍是复性过程中尿素依赖性聚集，导致酶不可逆失活。使用凝胶过滤高效液相色谱、荧光光谱和二硫键分析进行复性研究获得的信息，得出了一种最佳酶复性方案，该方案产生了高活性（即预期活性的65%-75%）且适度稳定的真菌合成酶/羟化酶。
