Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli.
Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli.
复制标题
双花扁豆种子凝集素在大肠杆菌中的表达和部分表征。
DOI：
10.1006/abbi.1994.1397
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发表时间：
1994
影响因子：
3.9
通讯作者：
Etzler,ME
中科院分区：
文献类型：
--
作者：
Chao,Q;Casalongue,C;Quinn,JM;Etzler,ME
利用pET表达载体在大肠杆菌中表达了豆科植物双花扁豆种子凝集素。用甲硫氨酸替换该凝集素的22个氨基酸的信号序列使凝集素表达水平增加超过100倍。约20%的表达的种子凝集素是可溶的，其余的溶解在8 M尿素和复性快速稀释。在可溶性和复性形式之间没有检测到物理化学性质或活性的差异。NH 2-末端氨基酸分析和免疫印迹，使用抗体识别的COOH-末端的只有nontruncated亚基的天然异源低聚体，建立了表达的凝集素具有一级结构相当于亚基I的天然种子凝集素。所表达的种子凝集素是有活性的，如其结合血型A + H物质-琼脂糖凝胶的能力所证明的，并且用N-乙酰半乳糖胺从该柱中特异性洗脱。然而，使用灵敏的ELISA比较表达的凝集素与天然种子凝集素的活性显示，表达的凝集素对血型A + H物质的亲和力略低于天然种子凝集素。通过分子排阻色谱测定，表达的凝集素的Mr也低于种子凝集素。
The seed lectin from the legume,Dolichos biflorus, was expressed inEscherichia coliusing the pET expression vector. Replacement of the 22-amino acid signal sequence of this lectin with a methionine increased the level of lectin expression greater than 100-fold. Approximately 20% of the expressed seed lectin was soluble; the remainder was solubilized in 8 M urea and renatured by rapid dilution. No difference in physicochemical properties or activity was detected between the soluble and renatured forms. NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontruncated subunit of the native heteroligomer, established that the expressed lectin has a primary structure equivalent to subunit I of the native seed lectin. The expressed seed lectin is active as evidenced by its ability to bind to blood group A + H substance-Sepharose and to be specifically eluted from this column withN-acetylgalactosamine. However, a comparison of the activity of the expressed lectin with the native seed lectin using a sensitive ELISA showed that the expressed lectin has a slightly lower affinity for blood group A + H substance than the native seed lectin. The expressed lectin also has a lowerMrthan the seed lectin as determined by molecular exclusion chromatography.