Production of lectins having unique carbohydrate-binding specificity by use of protein engineering technique

利用蛋白质工程技术生产具有独特碳水化合物结合特异性的凝集素

• 批准号: 04670138
• 负责人: YAMAMOTO Kazuo
• 金额: $ 1.34万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670138/
• 关键词: lectin; pharge; chimeric protein; sugar specificity; 糖鎖; 相互作用; 特異性; キメラ

The carbohydrate-binding domain of Bauhinia purpurea lectin (BPA), was investigated from Asp-N endoproteinase digests of BPA.A peptide which interacted with lactose was purified by means of lactose-Sepharose column chromatography. It consisted of 9 amino acids. Subsequently, we constructed a chimeric lectin gene by using a cDNA coding BPA in which nonapeptide sequence was replaced by the corresponding region of the mannose-binding Lens culinaris lectin. The chimeric lectin expressed in E.coli was found to bind mannosyl-bovine serum albumin and this binding was inhibitied by mannose. Then nonapeptide sequence was replaced with random amino acid sequences, which were introduced into cDNA coding BPA by use of polymerase chain reaction technique. The constructed lectin cDNA was inserted into lambda gtll or lambda foo expression vector, followed by expression in E.coli cells. In lambda gtll system, lectin was detected as fusion protein of beta-galactosidase. In lambda foo pharge, lectin molecule had not been detected yet.

利用紫荆凝集素（BPA）的Asp-N内源性蛋白酶酶切研究了其碳水化合物结合结构域。采用乳糖- sepharose柱层析法纯化了一种与乳糖相互作用的肽。它由9个氨基酸组成。随后，我们利用编码BPA的cDNA构建了一个嵌合凝集素基因，其中非肽序列被甘露糖结合Lens culinaris凝集素的相应区域所取代。在大肠杆菌中表达的嵌合凝集素能与甘露糖基-牛血清白蛋白结合，这种结合被甘露糖抑制。然后用随机氨基酸序列替换非肽序列，利用聚合酶链反应技术将其引入编码BPA的cDNA中。将构建的凝集素cDNA分别插入lambda gtll或lambda foo表达载体，在大肠杆菌细胞中表达。在lambda gtll系统中，凝集素被检测为β -半乳糖苷酶的融合蛋白。在lambda foo噬菌体中尚未检测到凝集素分子。

项目成果

Y.Konami, C.Ishida, K.Yamamoto, T.Osawa, & T.Irimura: "A unique amino acid sequence involved in the putative carbohydrate-binding domain of sialic acid specific legume lectin." J.Biochem.(in press). (1994)

Y.Konami、C.Ishida、K.Yamamoto、T.Osawa、

K.Yamamoto: "Lectins : Structures and lectin-sugar interactions." Membrane. 19. 11-18 (1994)

K.Yamamoto：“凝集素：结构和凝集素-糖相互作用。”

K.Yamamoto: "cDNA cloning and expression of plant lectins from legmenous family" Lectins and Glycobiology. 469-473 (1993)

K.Yamamoto：“豆科植物凝集素的 cDNA 克隆和表达”凝集素和糖生物学。

山本 一夫: "Carbohydrate-binding peptides from several anti-H(0) lectins." J.Biochem.111. 436-439 (1992)

Kazuo Yamamoto：“来自几种抗 H(0) 凝集素的碳水化合物结合肽。”J.Biochem.111（1992）。

K.Yamamoto: "レクチンの糖鎖認識における構造論的解析" 膜. 19. 11-18 (1994)

K. Yamamoto：“凝集素糖链识别的结构分析”膜。19. 11-18 (1994)