CATALYTIC ANTIBODIES FOR PEPTIDE SYNTHESIS

用于肽合成的催化抗体

基本信息

批准号：
2608926
负责人：
RALPH F HIRSCHMANN
金额：
$ 22.6万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-01-01 至 1998-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2608926
关键词：
X ray crystallography abzyme chemical condensation computer simulation peptide chemical synthesis site directed mutagenesis

项目摘要

DESCRIPTION: This research program represents a collaboration between Professor Hirschmann, Smith and Christianson at the University of Pennsylvania and Professor Benkovic at the Pennsylvania State University (Section 5). The principal goal of the program is to produce catalytic antibodies which will promote efficient condensation of peptide fragments. The use of such antibodies to effect peptide bond formation should manifest the advantages of enzymatic peptide synthesis (i.e., minimal need for protecting groups, chemospecificity, and stereospecificity) and avoid the limitations associated with enzymes instability, enzyme-induced hydrolysis, and substrate related solubility problems. Importantly, one will not be restricted by the repertoire of available enzymes. These considerations lead us to propose the following specific aims for the 04-07 years of GM-45611: (1) To explore strategies for in situ activation of stable acyl fragments immediately prior to antibody-catalyzed couplings. To this end, the investigators will develop second-generation haptens that mimic tetrapeptides (i.e., transition limitations, and mechanism of catalysis by antibody 16G3 via: (a) studying a variety of substrates; (b) defining the kinetics of catalysis; and (c) determining the three-dimensional structures of Fab 16G3, both uncomplexed and complexed with a transition state analog and with substrates. (3) To develop antibodies capable of catalyzing peptide formation from inactivated acyl donors and larger fragments, the investigators will perform site-specific mutagenesis on antibody 16G3. (4) To prepare large amounts of 16G3 and its single-chain analog, as required for subgoals 2 and 3. They have already begun to generate 16G3 on a large scale (ca. 1-34g) at the Penn Cell Center. (5) In addition, the investigators propose to: (a) prepare FAb (or Fab') fragments of 16G3 by routine proteolysis; and (b) convert the hybridoma-cell RNA to single-chain antibodies (SCA). (6) To exploit the newly discovered phosphonyl trialkylammonium salts, refining and further developing our effective protocol for the synthesis of phosphonate esters and phosphonamides. Assuming that the concept of using catalytic antibodies in fragment condensation is viable, and that it can be applied to the coupling of longer fragments, condensation is, a suitable leap of faith would be to attempt to use a catalytic antibody to couple an appropriately C-terminally derovatozed S-peptide to the corresponding S-protein, both readily available from ribonuclease A. The reconstruction of ribonuclease A from its subtilisin degradation products would be a dramatic demonstration of the scope of this approach. This has previously been achieved enzymatically. The investigators will then apply catalytic antibodies to couple fragments of other known proteins and subsequently of peptides and proteins that incorporate uncoded amino acids.

描述：该研究计划代表赫希曼教授，史密斯和克里斯蒂安森宾夕法尼亚州宾夕法尼亚州立大学的本科维奇教授（第5节）。该计划的主要目标是产生催化将促进肽有效凝结的抗体碎片。使用这种抗体作用形成肽键应表现出酶促肽合成的优势（即保护群体，化学特异性和立体特定）并避免与酶有关的局限性不稳定性，酶诱导的水解和底物相关溶解度问题。重要的是，一个人不会受到曲目的限制可用的酶。这些考虑使我们提出了遵循GM-45611 04-07年的特定目的：（1）探索立即实现稳定酰基碎片的原位激活的策略在抗体催化耦合之前。为此，调查人员将形成模拟四肽的第二代触觉（即抗体16G3催化的过渡限制和机制通过：（a）研究各种底物；（b）定义动力学催化; （c）确定晶圆厂的三维结构 16G3，既没有复杂又与过渡态类似物和与底物。（3）开发能够催化的抗体来自灭活的酰基供体和较大片段的肽形成，研究人员将对抗体16G3进行位点特异性诱变。（4）准备大量16G3及其单链类似物子目标2和3所必需的。它们已经开始产生16G3 在宾夕法尼亚州牢房中心大规模（约1-34G）。（5）此外，调查人员建议：（a）准备Fab（或Fab'）碎片 16G3通过常规蛋白水解；（b）将杂交瘤细胞RNA转换为单链抗体（SCA）。（6）利用新发现的磷酸试验kymonium盐，精炼并进一步发展我们的合成膦酸酯和的有效方案膦酰胺。假设使用催化抗体的概念在碎片中，凝结是可行的，可以将其应用于较长碎片的耦合，凝结是一个合适的信仰飞跃将尝试使用催化抗体夫妇适当的C末端降低的S肽与相应的 S蛋白，都可以从核糖核酸酶A中获得。核糖核酸酶从其枯作蛋白降解的重建产品将是对此范围的戏剧性演示方法。以前已通过酶促实现这一目标。这然后，调查人员将催化抗体对几个片段其他已知蛋白质以及随后的肽和蛋白质结合未编码的氨基酸。