Prak Krisna, Maruyama Yukie, Maruyama Nobuyuki, Utsumi Shigeru
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Peptides. 2006 Jun;27(6):1179-86. doi: 10.1016/j.peptides.2005.11.007. Epub 2005 Dec 13.
The peptide IIAEK derived from beta-lactoglobulin has a hypocholesterolemic activity greater than that of beta-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.
源自β-乳球蛋白的肽IIAEK具有比β-谷甾醇更强的降胆固醇活性。为了创建具有多个这种有价值肽序列拷贝的食品蛋白，我们将编码该肽的核苷酸序列串联多聚体引入到与大豆球蛋白A1aB1b亚基的五个可变区相对应的DNA区域，并在大肠杆菌中表达这些突变体。比较了五个突变体的表达水平和溶解性，每个突变体在每个可变区都含有四个IIAEK序列。总体而言，可变区IV和V中含有四个IIAEK序列的突变体的表达水平和溶解性最佳，其次是II > III > I。此外，在可变区IV引入第五个IIAEK序列并没有降低表达水平和溶解性。将IIAEK的数量增加到7和10会略微降低表达水平，而它们的溶解性分别降至40%和1%。将各种突变组合以获得含有尽可能多IIAEK序列的突变体。一些所得突变体以可溶形式表达。来自可变区IV和V组合（IV-4 + V-4）的含有八个IIAEK的突变体在表达水平和溶解性之间表现出最佳平衡，其次是可变区II和III的组合（II-4 + III-4）。这些突变体的可溶部分通过疏水、凝胶过滤和离子交换柱色谱法进行纯化。用胰蛋白酶体外消化这两个突变体释放的IIAEK肽的产量约为80%。这是首次报道可以将大量生理活性肽引入食品蛋白。
