Subtilisin-like Plant Protease Cleaving at Multiple Acidic Amino Acid Residues

9722984 Tan-Wilson Protease C1在生长早期催化主要大豆储存球蛋白的初始有限蛋白分解。它的部分基因序列表明它属于丝氨酸蛋白酶的枯草杆菌蛋白家族。这一点值得进一步研究，因为在这个大家族中，只有7种酶来自植物。此外，蛋白酶C1‘S裂解位点的序列显示出对P1、Pl’和P4‘位的谷氨酸或天冬氨酸残基的独特需求。蛋白酶C1的切割特异性反映了与枯草杆菌蛋白相关的Kex2-1ike蛋白加工酶的特异性，只是后者需要多个碱性氨基酸残基，而不是多个酸性氨基酸残基。在这个项目中，将通过检测植物不同发育阶段的不同部位的活性和通过将寡核苷酸探针与Northern blotts杂交来研究蛋白质和合成肽底物的基因表达，从而进一步描述蛋白酶C1的切割特异性。该项目还将确定储存带有Glu和Asp残留物串的种子蛋白质的其他豆科物种是否具有类似于蛋白酶C1的酶，以在萌发过程中启动蛋白质分解。将寡核苷酸探针与Southern blotts杂交将揭示这些物种中是否存在类似蛋白水解酶的基因。如果呈阳性，将对这些植物的成熟种子和子叶进行类蛋白酶活性的研究和寡核苷酸探针与Northern blotts的杂交。有关这种蛋白水解酶的信息将对试图克隆豆类贮藏蛋白中具有商业重要性的产品的研究小组感兴趣，并将有助于我们理解酶的进化。豆类是蛋白质和食用油的重要来源。当这些植物的遗传转化变得更容易时，储存蛋白很可能成为操纵以改善营养质量或生产治疗性多肽的目标。在后一种情况下，了解这些蛋白质将遇到的蛋白水解酶的切割特性将是至关重要的，因为这些酶切割蛋白质，从而破坏预期的基因产品。本项目是对蛋白水解酶C1的研究，它是大豆中的一种蛋白水解酶，在大豆萌发和幼苗早期生长过程中启动储存蛋白质的分解。该项目的目的是确定该酶在裂解部位需要什么特征，以及在植物的生长周期中该酶何时以活性形式存在，以便能够设计出避免其作用的克隆策略。可替换地，在生物技术的蛋白质产品加工中，可以很好地利用蛋白质水解酶，例如大豆蛋白水解酶C1，它在清除位置方面具有很强的区分性。

9722984 Tan-Wilson Protease C1 catalyzes the initial limited proteolysis of the major soy storage globulins during early growth. Its partial cDNA sequence suggests that it belongs to the subtilisin family of serine proteases. This merits further study since only seven of the enzymes in this large family of proteases are from plants. Moreover, sequences at protease C1's cleavage sites show a unique requirement for glutamate or aspartate residues at the P1, Pl', and P4' positions. The cleavage specificity of protease C1 mirrors the specificity of the subtilisin-related Kex2-1ike protein-processing proteases, except that the latter require multiple basic instead of multiple acidic amino acid residues. In this project, the cleavage specificity of protease C1 will be further delineated using protein and synthetic peptide substrates gene expression will be studied by examining different parts of the plant at different developmental stages for activity and by hybridization of oligonucleotide probes to northern blots. The project would also determine whether other legume species that store seed proteins bearing strings of Glu and Asp residues have protease C1-like enzymes to initiate proteolysis during germination. Hybridization of oligonucleotide probes to Southern blots would reveal whether protease Cl-like genes exist in these species. If positive, maturing seeds and seedling cotyledons of these plants would be studied for protease Cl-like activity and hybridization of oligonucleotide probes to northern blots. Information on this proteolytic enzyme would be of interest to research groups attempting to clone commercially important products in legume storage proteins, and would contribute to our understanding of enzyme evolution. Legumes constitute important sources of protein and edible oil. When genetic transformation of these plants becomes easier, the storage proteins are likely targets for manipulation to improve nutritional quality or to produce therapeutic peptides. In the latter case, it will be vital to know the cleavage specificities of the proteolytic enzymes that these proteins would encounter since these enzymes cleave proteins, thus destroying the intended gene product. This project is a study of protease C1, a proteolytic enzyme in soybean that initiates the breakdown of the stored proteins during soybean germination and early seedling growth. The aims of the project are to determine what features the enzyme requires in a cleavage site and where in the plant and when during the plant's growth cycle this enzyme is present in active form so that cloning strategies to avoid its action can be designed. Alternatively, proteolytic enzymes such as soybean protease C1 that is very discriminating with regard to clevage site may be put to good use in processing protein products for biotechnology.