Patel Girijesh K, Gupta Amit K, Gupta Akshita, Mishra Manisha, Singh Pradhyumna K, Saxena Anil K, Sharma Ashwani K
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, India.
Protein Pept Lett. 2014;21(2):108-14. doi: 10.2174/09298665113209990090.
A thermotolerant protein with trypsin inhibitory activity designated as CaTI was purified to homogeneity from seeds of Cassia absus. Gel filtration chromatography and SDS-PAGE analysis showed the apparent molecular mass of ~20 kDa. Partial internal sequences indicate that CaTI belongs to Kunitz-inhibitor family. CaTI inhibits the bovine trypsin in 1:1 molar ratio and exhibited a competitive-type inhibitory activity with Ki = 5.6 x 10(-9) M. The inhibitory activity was retained over a broad pH range (2-12). Thermal stability study showed that it is stable up to 80 °C and inhibition activity reduced at and above 90 °C which might be due to the presence of predominantly β-sheets revealed by the CD study. The proteolysis studies of CaTI exhibited strong resistance to proteolysis by different proteases tested. The studies show that CaTI can be used as potential candidates for the development of the transgenic plant against the microbes and insect pests.
从望江南种子中纯化出一种具有胰蛋白酶抑制活性的耐热蛋白，命名为CaTI，达到了同质纯。凝胶过滤色谱法和SDS-PAGE分析表明其表观分子量约为20 kDa。部分内部序列表明CaTI属于库尼茨抑制剂家族。CaTI以1:1的摩尔比抑制牛胰蛋白酶，并表现出竞争性抑制活性，抑制常数Ki = 5.6 x 10(-9) M。其抑制活性在较宽的pH范围（2 - 12）内保持稳定。热稳定性研究表明，它在高达80°C时稳定，在90°C及以上抑制活性降低，这可能是由于圆二色性研究显示其主要存在β-折叠结构。对CaTI的蛋白水解研究表明，它对所测试的不同蛋白酶具有很强的抗蛋白水解能力。这些研究表明，CaTI可作为开发抗微生物和害虫转基因植物的潜在候选物。
