Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest – Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ± 11 nM) and chymotrypsin (Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC₅₀ = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC₅₀ = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

从木豆的野生近缘种Rhynchosia sublobata的种子中提取的粗蛋白酶抑制剂（CPIs）对鳞翅目昆虫害虫– Achaea janata的幼虫肠胰蛋白酶样蛋白酶具有明显的抑制活性。因此，鲍曼-Birk抑制剂基因的全长cDNA（RsBBI1）从的未成熟种子克隆R. sublobata。它包含一个360 bp的ORF，编码一个119个氨基酸的多肽（13.3 kDa）链，其N端信号序列包含22个氨基酸。氨基酸序列和系统发育分析共同表明，RsBBI1与大豆和菜豆的BBI密切相关。对应于RsBBI1的cDNA序列成熟蛋白（89个氨基酸延伸）在大肠杆菌中表达。使用胰蛋白酶亲和层析纯化分子量为9.97 kDa的重组rRsBBI1蛋白。纯化的rRsBBI1对牛胰蛋白酶（Ki为358±11 nM）和胰凝乳蛋白酶（Ki为446±9 nM）均表现出非竞争性抑制作用。它对这些蛋白酶的抑制活性在高温（> 95°C）和宽pH范围内稳定，但对二硫苏糖醇（DTT）的还原敏感，表明二硫键在显示其活性方面的重要性。此外，rRsBBI1显示显著抑制活性（IC ₅₀  = 70毫微克）上A.贾纳塔幼虫肠胰蛋白酶样蛋白酶（AjGPs）。相反，与对AjGPs相比，它对棉铃虫幼虫肠胰蛋白酶样蛋白酶（HaGPs）的抑制活性<1％（IC ₅₀  = 8μg）。此外，体内饲喂实验清楚地表明了rRsBBI1对A. janata幼虫生长和发育的有害作用，这表明可以进一步利用rRsBBI1的这种特性。