Stubbs M E, Carver J P, Dunn R J
J Biol Chem. 1986 May 15;261(14):6141-4.
In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.
为了探究豆科植物凝集素糖肽特异性的分子基础，我们开发了一个实验系统，在该系统中可以将特定的氨基酸改变引入豌豆凝集素的碳水化合物结合位点。这个系统基于在大肠杆菌中生产豌豆凝集素。编码凝集素的质粒由从豌豆种子中分离得到的两个凝集素cDNA序列构建而成（希金斯，T. J. V.，钱德勒，P. M.，祖拉夫斯基，G.，巴顿，S. C.，以及斯宾塞，D.（1983年）《生物化学杂志》258卷，9544 - 9549页），以及一个基于大肠杆菌外膜脂蛋白基因的表达载体（中村，K.，以及井上，M.（编）（1982年）《欧洲分子生物学组织杂志》1卷，771 - 775页）。凝集素以单条多肽链的形式产生，并在大肠杆菌细胞中形成不溶性聚集体（2 - 5毫克/升）。通过在盐酸胍中溶解聚集体、在氯化锰和氯化钙存在的情况下复性以及在葡聚糖凝胶上进行亲和纯化来回收有功能的凝集素。这个过程产生一种均一的28,000道尔顿的蛋白质。在血细胞凝集抑制试验中对重组凝集素和天然豌豆凝集素进行比较表明，这两种凝集素在碳水化合物结合特性方面没有可检测到的差异。
