Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Kwanak-Gu, Seoul, South Korea; Institute of Bioengineering, Seoul National University, Kwanak-Gu, Seoul, South Korea.
Biotechnol Bioeng. 2013 Nov;110(11):2865-73. doi: 10.1002/bit.24970. Epub 2013 Jul 10.
Since detailed evaluation of specific transglutaminases (TGs) from various species requires identification of their substrate specificities, rapid substrate screening method by measurement of their relative activities is in great demand. Here, a novel evaluation method of TG activity was developed using two recombinant fluorescent proteins (FPs), that is, eYFP and DsRed, tagged with TG substrate peptides. By cross-linking the two FPs based on the tagged target peptide sequences at their C-terminus, the expression of co-transformed TG allows quenching of the yellow fluorescence intensities. It was shown that the degree of in vivo fluorescent quenching by the TG activity agrees well with its in vitro reaction data, suggesting that this system can be used to identify relative substrate specificity of TGs for target peptide sequences. Using this method, the lysine substrates of TGs from Bacillus species (BTG) were evaluated, and the newly selected pentapeptide, KTKTN showed almost the same reactivity with the well-known hexa-lysine (K₆) substrate for BTG reaction.
由于需要鉴定不同物种的特定转谷氨酰胺酶（TGs）的底物特异性，因此非常需要一种通过测量相对活性来快速筛选底物的方法。这里，我们使用两种带有 TG 底物肽的重组荧光蛋白（FPs），即 eYFP 和 DsRed，开发了一种新的 TG 活性评估方法。通过在 C 末端的标记靶肽序列上交联两个 FP，共转化的 TG 的表达可以猝灭黄色荧光强度。结果表明，TG 活性的体内荧光猝灭程度与体外反应数据非常吻合，表明该系统可用于鉴定 TG 对靶肽序列的相对底物特异性。使用该方法，评估了芽孢杆菌属（BTG）的 TG 的赖氨酸底物，并且新选择的五肽 KTKTN 对 BTG 反应的熟知的六赖氨酸（K₆）底物表现出几乎相同的反应性。
