Trumbly R J, Robbins P W, Belfort M, Ziegler F D, Maley F, Trimble R B
J Biol Chem. 1985 May 10;260(9):5683-90.
The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.
来自褶皱链霉菌的内切-β-N-乙酰氨基葡萄糖苷酶H（Endo H）基因已被克隆到大肠杆菌质粒pKC30中（岛武，H.，和罗森伯格，M.（1981年）《自然》272，128 - 132），从而使该基因的表达受强λ启动子pL的控制。构建体pKCE3包含来自乳糖操纵子的一个定位合适的大肠杆菌核糖体结合位点（罗宾斯，P.W.，特林布尔，R.B.，沃思，D.F.，赫林，C.，马利，F.，马利，G.F.，达斯，R.，吉布森，B.W.，和比曼，K.（1984年）《生物化学杂志》259，7577 - 7583），用于转化对含有温度敏感阻遏物的λ原噬菌体溶原化的大肠杆菌菌株。通过将pKCE3溶原菌培养物转移至42℃，Endo H的产生开始，并在约1小时内呈线性。酶产量比从褶皱链霉菌的可比培养物中获得的产量放大了150倍，占细胞总蛋白的3%至4%，这使得能够通过快速的四步程序将Endo H纯化至同质。尽管大多数克隆的Endo H被大肠杆菌分泌到周质空间中，但其4 kDa的前导序列肽（罗宾斯等人（1984年））在加工过程中仅被部分去除。结果，纯化的pKCE3 Endo H是分子的异质群体，平均分子量为31 kDa，而褶皱链霉菌正常分泌的完全加工产物的分子量为28.9 kDa。尽管克隆的pKCE3产物上残留约2 kDa的前导序列，但从大肠杆菌或褶皱链霉菌纯化的酶在底物特异性或稳定性特征方面均未检测到差异。对于糖蛋白研究特别有价值的是发现基因工程改造后的Endo H完全没有蛋白水解污染物。
