High-throughput profiling of proteases with phage and arrays

蛋白酶是真核细胞中一类重要的酶， 在细胞生长，发育，细胞信号传导， 凋亡和自噬。然而，调控不当会导致疾病，例如癌症 细胞通常分泌金属蛋白酶，降解细胞外基质， 细胞转移并在身体的各个部位扎根。蛋白酶的工作原理- 在其底物中形成线性区域，通常为6至8个残基，并水解肽键， 从而将它们的底物片段化为N-末端和C-末端片段。理解 被蛋白酶切割的肽序列的范围，称为“蛋白水解特征”或 “底物概况”，具有许多科学意义和实际效益。一个强大的方法 用于确定重组蛋白酶的蛋白水解特征或底物谱的方法 通过使用组合肽库。在这项努力中，有两个具体目标。在 目的#1，我们将开发一种改进的噬菌体载体，用于蛋白酶位点鉴定， 更有效地切割识别序列，更均匀地表示氨基酸， 和鉴定更长的识别序列（高达10个AA）。载体将携带 卡那霉素抗性基因和基因III的第二个拷贝，所述基因III在N-末端用 AviTag并在三个不同位置具有相同的克隆位点盒。在向量之后 已证明适合作为两种模型蛋白酶、寡核苷酸 将以19个三核苷酸亚磷酰胺的10聚体区域随机排列， 频率编码19个氨基酸（不包括半胱氨酸），用于克隆到每个位点和大（> 1010）图书馆创建。细菌菌株XL 1-Blue中的OmpT基因将被敲除， 同源重组以消除文库中噬菌体展示肽的损失， 带有二元残基。在目标#2中，Tango将使用两个工具来评估新库的实用性。 可商购的蛋白酶、人金属蛋白酶、ADAM 15和埃希氏菌属蛋白酶。 coli蛋白酶DegP.病毒体将被捕获在中性抗生物素蛋白包被的磁珠上， 用每种蛋白酶，并且输出将通过下一代测序（NGS）进行测序。 然后，Nimble Therapeutics将在阵列上合成200万种肽，相当于10或100万种肽。 NGS发现的每个肽序列的拷贝数更多，沿着系统氨基酸 替换在所选肽中鉴定的任何基序。这些肽将携带一个 在它们的C-末端具有与荧光链霉亲和素结合的肽标签，并且在它们的C-末端具有PEG接头。 N-末端，以使它们远离阵列表面。阵列将与亲- 梳理，与荧光链霉亲和素孵育，扫描将揭示哪些斑点失去荧光- 由于蛋白质分解而引起的。为了证实这些肽是它们各自的真正底物， Tango将为每种蛋白酶订购20个合成肽，并通过以下方式确定切割位点： 质谱分析法来成功完成拟议的实验将创造巨大的价值。 Tango将建立专有的噬菌体文库，并完善标准操作方案（SOP）， 其可以确定任何客户蛋白酶的底物分布。未来的第二阶段SBIR适用于- 阳离子将使Tango能够定义50种人类和病原体蛋白酶的谱， 为开发肽模拟物抑制剂以治疗人类 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.