Chimeric enzyme for host nucleic acid autohydrolysis

用于宿主核酸自动水解的嵌合酶

基本信息

批准号：
6833053
负责人：
James Williams
金额：
$ 14.38万
依托单位：
NATURE TECHNOLOGY CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-23 至 2005-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6833053
关键词：
Escherichia coli biotechnology chimeric proteins deoxyribonuclease I endopeptidases endoribonucleases fusion gene gene expression genetic strain high throughput technology hybrid enzyme hydrolysis nucleic acid chemical synthesis nucleic acid purification plasmids polymerase chain reaction protein engineering

项目摘要

DESCRIPTION (provided by applicant): DNA vaccines and gene medicines are emerging as an important new class of pharmaceuticals that are derived from bacterial plasmids. For use in humans, plasmid DNA should be essentially free from bacterial genomic DNA and RNA, protein and endotoxin. The hypothesis is that E. coli plasmid DNA can be more efficiently purified by co-expressing RNase and DNase in plasmid producing strains of bacteria. The strategy will be to make hybrid proteins (expressing RNase A and phage T5 DNA exonuclease) and secrete them into the periplasmic space, releasing them at the appropriate time to allow digestion of E. coli RNA and genomic DNA. In Specific Aim I, we will construct recombinant genes for expression of RNase and T5 exonuclease. In Specific Aim II, we will investigate the use of the recombinant enzymes in the purification of plasmids. The overall goal of the proposed Phase I feasibility study is to determine whether such a bacterial strain is a significant advantage in the purification of plasmid DNA. If successful, Phase II studies will include improved constructs for insertion of the genes into the E. coli chromosome; creating the strains needed; development of a third enzyme (protease); and development of an inducible system for introducing the foreign proteins into the cytoplasm during the final hours of fermentation. In Phase Ill, NTC will introduce the improved strains in commercial plasmid production. Success in constructing one or more useful strains of bacteria expressing RNAse, DNAse and/or protease enzymes in a controllable fashion (milestone II) would be a significant development for DNA purification on a commercial scale. Such strains will be rapidly introduced into NTC's large scale DNA manufacturing process, and will also be made available for licensing to other commercial and academic users through the company's technology transfer and licensing program. Currently, experimental gene drugs and DNA vaccines are routinely made on the gram scale, mostly for use in animal safety and efficacy studies. However, the advent of FDA approved commercial products will require scaling up to kilograms, for example, for a widely used vaccine. Judging from the current price of $22,000- $46,000/gram of DNA produced, a less costly, scalable fermentation process is badly needed. The economic benefits and potential market size for the resulting products can be readily appreciated. The ability to use endogenously produced recombinant enzymes would be a significant advantage (in terms of safety and economy) over the use of animal-derived or exogenously added enzymes. The likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.

描述（由申请人提供）： DNA疫苗和基因药物正在成为一种重要的新型药物，这些药物源自细菌质粒。为了在人类中使用，质粒DNA应基本不含细菌基因组DNA和RNA，蛋白质和内毒素。假设是，大肠杆菌质粒DNA可以通过在质粒产生细菌菌株中共表达RNase和dNase来更有效地纯化。该策略将是使杂化蛋白（表达RNase A和Phage T5 DNA外切核酸酶）并将其分泌到周质空间中，并在适当的时间释放它们以消化大肠杆菌RNA和基因组DNA。在特定的目标I中，我们将构建用于表达RNase和T5外切核酸酶的重组基因。在特定的目标II中，我们将研究重组酶在质粒纯化中的使用。提出的I期可行性研究的总体目标是确定这种细菌菌株在纯化质粒DNA的纯化方面是否是一个重要的优势。如果成功，第二阶段的研究将包括改进的构造，将基因插入大肠杆菌染色体中。产生所需的菌株；开发第三酶（蛋白酶）；以及在发酵的最后几个小时内将诱导系统引入细胞质中的诱导系统。在阶段生病时，NTC将引入商业质粒生产中改善的菌株。以可控制的方式（Milestone II）构建一种或多种有用的细菌，表达RNase，DNase和/或蛋白酶的细菌菌株的成功将是商业尺度上DNA纯化的重大发展。此类菌株将迅速引入NTC的大规模DNA制造过程中，还将通过公司的技术转移和许可计划获得许可。目前，实验性基因药物和DNA疫苗通常是按照量表进行的，主要用于动物安全和功效研究。但是，FDA批准的商业产品的出现将需要扩展多达公斤，例如，使用广泛使用的疫苗。从目前的价格为$ 22,000- $ 46,000/克的DNA的价格来看，急需成本较低，可扩展的发酵过程。可以很容易地赞赏所得产品的经济利益和潜在的市场规模。在使用动物来源或外源添加的酶中，使用内源性重组酶的能力将是一个重要的优势（在安全和经济方面）。拟议的工作产生的商业有用和有价值的产品的可能性被认为很高。