Chen L J, Ford C, Kusnadi A, Nikolov Z L
Department of Genetics, Iowa State University, Ames 50011.
Biotechnol Prog. 1991 May-Jun;7(3):225-9. doi: 10.1021/bp00009a004.
We have previously shown (Chen et al., 1991) that a beta-galactosidase (beta-gal) fusion protein (BSB133) containing 133 amino acids (aa) from the C-terminus of Aspergillus glucoamylase (GA) adsorbs strongly to starch compared to beta-gal, due to the presence of the GA starch-binding domain. We have now made deletions at the N-terminus of this 133-aa region to test the minimal size required for starch binding of beta-gal fusion proteins. Three fusion proteins (BSB119, BSB103, and BSB80) were genetically engineered, containing 119, 103, and 80 C-terminal aa from GA, respectively. The fusion proteins were expressed in Escherichia coli and purified. Purified BSB119 adsorbed to native starch at least 2-fold more strongly than did BSB133 or fusion proteins with shorter tails. Adsorption isotherms generated over a wide range of initial concentrations indicated a 10-fold difference in the loading capacity of starch for BSB119 (36.5 mg of protein/g of starch) compared to beta-gal (3.7 mg of protein/g of starch). Adsorption constants calculated from the initial slopes of the isotherms indicated a nearly 30-fold difference in affinity to starch for BSB119 (Kad = 63 mL/g of starch) compared to beta-gal (Kad = 2.3 mL/g of starch). BSB119 in the presence of crude enzyme extracts also bound to starch with a high affinity compared to a beta-gal control. Potential applications of the starch-binding tail include enzyme immobilization to starch or recovery and purification of target proteins from crude extracts.
我们之前已经表明（Chen等人，1991年），与β-半乳糖苷酶（β-gal）相比，一种包含来自米曲霉糖化酶（GA）C末端133个氨基酸（aa）的β-半乳糖苷酶（β-gal）融合蛋白（BSB133）由于存在GA淀粉结合结构域，能与淀粉强烈结合。我们现在对这个133个氨基酸区域的N末端进行了缺失，以测试β-半乳糖苷酶融合蛋白与淀粉结合所需的最小尺寸。通过基因工程构建了三种融合蛋白（BSB119、BSB103和BSB80），分别包含来自GA的119、103和80个C末端氨基酸。这些融合蛋白在大肠杆菌中表达并纯化。纯化后的BSB119与天然淀粉的结合能力至少比BSB133或尾巴更短的融合蛋白强2倍。在广泛的初始浓度范围内生成的吸附等温线表明，与β-gal（3.7 mg蛋白质/g淀粉）相比，淀粉对BSB119的负载能力有10倍的差异（36.5 mg蛋白质/g淀粉）。根据等温线的初始斜率计算出的吸附常数表明，与β-gal（Kad = 2.3 mL/g淀粉）相比，BSB119对淀粉的亲和力有近30倍的差异（Kad = 63 mL/g淀粉）。与β-gal对照相比，在粗酶提取物存在的情况下，BSB119也能与淀粉高亲和力结合。淀粉结合尾巴的潜在应用包括将酶固定在淀粉上或从粗提取物中回收和纯化目标蛋白。
