High-throughput profiling of proteases with phage and arrays

在所有真核细胞中，蛋白水解酶代表着一类重要的酶。 在细胞生长、发育、细胞信号转导过程中激活和破坏蛋白质的关键作用， 细胞凋亡和自噬。然而，错误的监管可能会导致疾病，例如癌症 细胞通常会分泌金属蛋白酶，降解细胞外基质，从而允许癌症发生。 细胞转移并在体内不同位置扎根。蛋白水解酶的作用是通过识别- 在它们的底物中形成线状区域，通常是6到8个残基，并水解肽键， 从而将它们的底物碎裂成N-末端和C-末端片段。理解 被一种蛋白水解酶切割的一系列多肽序列，称为“蛋白分解信号”或 “底物剖面”，具有许多科学意义和实际效益。一种强大的方法 为了确定重组酶的蛋白水解性特征或底物分布， 通过使用组合多肽文库。在这一努力中，有两个具体目标。在……里面 目的#1，我们将开发一种改进的噬菌体载体，通过允许 更有效地切割识别的序列，更均匀地表示氨基酸， 和识别更长的识别序列(最多10个氨基酸)。载体将携带 卡那霉素抗性基因和N端标记的基因III的第二个拷贝 AviTag和在三个不同位置有相同的克隆站点盒式磁带。在向量之后 已被证明适合作为两种模型蛋白酶的底物，即寡核苷酸 将与19个三核苷酸亚磷酰胺随机排列的10聚体区域 编码19个氨基酸(不包括半胱氨酸)的频率用于克隆到每个位点和大的(&gt； 1010)创建的库。细菌XL1-Blue中的OmpT基因将被敲除 同源重组以消除噬菌体展示肽在文库中的丢失 携带二元残基。在第二个目标中，Tango将用两个库来评估新库的效用 商业上可获得的蛋白酶，人金属蛋白酶，ADAM15和大肠杆菌 Coli蛋白水解酶DegP.病毒粒子将被中性亲和素包裹的磁珠捕获，释放 对每一种蛋白酶，其输出将通过下一代测序(NGS)进行测序。 然后，Nimble Treateutics将在阵列上合成200万个肽，对应于10个或 NGS发现的每个肽序列的更多副本，以及系统氨基酸 在所选择的多肽中确定的任何基序的替换。多肽将携带一种 C末端的肽标签与荧光链霉亲和素结合，其C末端有一个聚乙二醇接头 N-端子，使其远离阵列表面。阵列将与专业- 与荧光链霉亲和素孵育的TEASE，扫描将发现哪些斑点失去了荧光- 这是由于蛋白质分解造成的。以确认这些多肽是它们各自的真实底物 Protease，Tango将为每个合成肽订购20个合成肽，并通过以下方式确定切割位置 质谱学。成功完成拟议的实验将创造巨大的价值。 Tango将构建专有的噬菌体文库并改进标准操作协议(SOP) 它可以确定任何客户蛋白酶的底物分布。未来的第二阶段SBIR应用-- 阳离子将使Tango能够定义50种人类和病原体蛋白水解酶，这将 为开发用于治疗人类的肽类抑制剂创造许可机会 疾病和感染。

Proteases represent an important class of enzymes in all eukaryotic cells where they play critical roles in activating and destroying proteins during cell growth, development, cell signaling, apoptosis, and autophagy. Misregulation, however, can lead to disease; for example, cancer cells often secrete metalloproteases that degrade the extracellular matrix, permitting cancers cells to metastasize and take root at various locations in the body. Proteases work by recogniz- ing linear regions, typically 6 to 8 residues, in their substrates and hydrolyzing a peptide bond, thereby fragmenting their substrate into N-terminal and C-terminal fragments. Understanding the range of peptide sequences cleaved by a protease, termed the "proteolysis signature" or "substrate profile", has many scientific implications and practical benefits. One powerful method for determining the proteolysis signature or substrate profile of a recombinant protease is through the use of combinatorial peptide libraries. In this effort, there are two specific aims. In Aim #1, we will develop an improved phage vector for protease site identification by allowing for more efficient cleavage of recognized sequences, more even representation of amino acids, and identification of longer recognition sequences (up to 10 AA).The vectors will carry the kanamycin resistance gene and a second copy of gene III that is tagged at the N-terminus with the AviTag and has the same cloning site cassette at three different locations. After the vectors have been demonstrated to be suitable as substrates for two model proteases, oligonucleotides will be ordered with a 10-mer region randomized with 19 trinucleotide phosphoramidites at equal frequency encoding 19 amino acids (excluding cysteine) for cloning into each site and large (> 1010) libraries created. The OmpT gene in the bacterial strain, XL1-Blue, will be knocked out by homologous recombination to eliminate the loss of phage-displayed peptides in the libraries that carry dibasic residues. In Aim #2, Tango will evaluate the utility of the new libraries with two commercially available proteases, the human metalloprotease, ADAM15, and the Escherichia coli protease, DegP. Virions will be captured on neutravidin-coated magnetic beads, released with each protease, and the output will be sequenced by Next Generation Sequencing (NGS). Nimble Therapeutics will then synthesize two million peptides on arrays, corresponding to 10 or more copies of each peptide sequence discovered by NGS, along with systematic amino acid replacements of any motifs identified among the selected peptides. The peptides will carry a peptide tag at their C-termini, which binds to fluorescent streptavidin, and a PEG linker at their N-termini to distance them it away from the array surface. Arrays will be incubated with the pro- tease, incubated with fluorescent streptavidin, and scans will reveal which spots lose fluores- cence due to proteolysis. To confirm that such peptides are true substrates for their respective protease, Tango will order 20 synthetic peptides for each and determine the cleavage site by mass spectrometry. Successful completion of the proposed experiments will create great value. Tango will build proprietary phage libraries and refine a standard operating protocol (SOP) with which it can determine the substrate profile for any client protease. A future Phase II SBIR appli- cation would enable Tango to define the profile 50 human and pathogen proteases, which would create licensing opportunities for the development of peptidomimetic inhibitors to treat human disease and infection.