High Throughput Protein Production by Novel E. coli Expression-Secretion System

项目概述/摘要：在后基因组时代，利用克隆DNA序列生产蛋白质变得越来越重要。细菌全基因组序列、cdna序列和数以万计的预测哺乳动物基因序列的可用性，创造了为这些基因生产蛋白质以供进一步研究的需要。在许多可用的方法中，大肠杆菌细胞表达仍然是一个可行的选择，因为表达载体系统很好地理解，大肠杆菌生长和诱导的成本最小。然而，大肠杆菌的表达会出现一些困难，如合成蛋白的不溶性、降解和细胞毒性；这削弱了它作为生产大量蛋白质的通用表达协议的效用。在第一阶段，我们将开发一种新的大肠杆菌表达-分泌-纯化系统，该系统将绕过与传统大肠杆菌表达相关的困难。该系统利用已经表征的大肠杆菌溶血素（HylA）蛋白分泌途径，当融合到HylA蛋白的c端部分时，该途径已被证明可以有效地分泌蛋白质。大肠杆菌中发现的其他3种蛋白质，即溶血素B、D和TolC，通过形成跨越细胞壁内外的多亚基通道，促进溶血素A的分泌(1)。Hly B-D-TolC通道通过大肠杆菌细胞壁主动易位天然Hyla或其c端融合体，使这些蛋白在诱导表达后在培养基中积累。与传统的大肠杆菌胞内表达相比，Hyla融合表达系统在蛋白质生产方面具有几个优势。这些优点包括：a)它避免了包涵体的形成，包涵体需要随后的溶解和恢复；b)分泌避免了毒性问题，因为分泌的蛋白质不会在细菌细胞中积累；c)完全分泌的蛋白质的纯化大大简化，因为蛋白质可以直接从澄清的生长介质中浓缩和纯化。已发表的研究表明，编码区如：scFv抗体链、大肠杆菌外膜孔蛋白（OmpF）、氯霉素乙酰转移酶（CAT）或l -天冬酰胺酶基因与Hly A基因的c端200个氨基酸部分融合，导致这些融合蛋白分泌到生长培养基中。我们将通过在待表达蛋白和c端Hyla结构域之间引入蛋白水解裂解位点来改进这一已发表的技术（1,4）。该蛋白水解位点将使我们能够在纯化过程中从感兴趣的蛋白中去除HylA蛋白序列。在I期之前，我公司从野生型大肠杆菌中克隆了溶血素分泌途径的4个关键基因，即：Hyl A、B、D和TolC蛋白。将HylB、HylD和TolC基因插入到设计用于在宿主DH5a大肠杆菌细胞中低水平表达这些通道形成蛋白的质粒中。HylA基因的c端结构域被克隆到pUC18质粒中，该质粒由6His标签和Hyl a c端组成。该载体使我们能够演示绿色荧光蛋白与HylA融合的表达和分泌，结果表明该方法可以分泌许多不同的蛋白质。该系统应该允许将标准化协议应用于几乎任何编码域的表达和纯化，因为我们测试用例中的表达不需要特定的诱导协议。此外，亲和纯化也大大简化了，因为在生长培养基中发现的大部分蛋白质都是由分泌蛋白组成的。我们的目标是开发一种适用于大量不同蛋白质的大肠杆菌表达-分泌-纯化的标准化系统。I期工作将包括：优化生长/诱导方案，在HylA和插入蛋白序列之间引入凝血酶蛋白水解识别序列；允许从分泌的蛋白质中去除hylA肽。将确定分泌所需的HylA的最小部分，并用该系统测试几个蛋白质编码区；包括一种有毒蛋白质。将对分泌蛋白的酶活性进行测量。项目描述：我们将以试剂盒、载体和细胞系的形式开发的产品将通过学术界、非营利组织、生物技术和制药公司，包括NIH支持的许多实验室，直接销售给40,000或更多的分子生物学和蛋白质组学实验室。MacConnell Research Corp.本身可以直接销售这些产品，因为我们已经为我们现有的产品建立了客户群。第二阶段产生的产品如果成功，每年的销售额将达到1000万至2000万美元。它们将提高蛋白质产量，产出高质量的蛋白质。这些产品将在第二阶段结束时基本准备好上市。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Protein production from cloned DNA sequences has become increasingly important in the post-genomic era. The availability of sequences from whole bacterial genomes, cDNAs, and tens of thousands of predicted mammalian genes have created the need to produce proteins for these genes for further studies. Of the many methods available, E. coli cell expression continues to be a viable choice as expression vector systems are well understood and the cost of E. coli growth and induction is minimal. E. coli expression, however, can present difficulties such as insolubility of synthesized protein, degradation, and cell toxicity; that detract from its utility as a universal expression protocol for production of a large number of proteins. In Phase I, we will develop a novel E. coli expression-secretion-purification system that will circumvent the difficulties associated with conventional E. coli expression. This system utilizes the already characterized E. coli hemolysin (HylA) protein secretion pathway that has been shown to allow efficient secretion of proteins when fused to the c-terminal portion of the HylA protein. 3 other proteins found in E. coli, namely hemolysin B, D and TolC facilitate the secretion of hemolysin A by forming a multi-subunit channel that spans the inner and outer cell wall (1). The Hly B-D-TolC channel actively translocates the native Hyla or its c-terminal fusions through the cell wall of the E. coli, so that these proteins accumulate in the media after induction of expression. The Hyla fusion expression system has several advantages for protein production over conventional intracellular expression in E. coli. These advantages include: a) it avoids the formation of inclusion bodies, which require subsequent dissolution and renaturation, b) secretion circumvents toxicity problems, as the secreted protein does not accumulate in the bacterial cells, and c) the purification of fully secreted proteins is greatly simplified, as the protein can be concentrated and purified directly from the clarified growth media. Published work has shown that fusion of coding regions such as: scFv antibody chain, E. coli outer membrane porin (OmpF), chloramphenicol acetyltransferase (CAT), or L-asparagenase genes to the c-terminal 200 amino acid portions of the Hly A gene results in secretion of these fusion proteins into the growth medium. We will improve this published technology by introducing a proteolytic cleavage site between the protein to be expressed and the c-terminal Hyla domain (1,4). This proteolytic site will enable us to remove the HylA protein sequence from the protein of interest during its purification. Prior to Phase I, our company cloned the 4 key genes of the hemolysin secretion pathway from wild type E. coli, namely the: Hyl A, B, D and TolC proteins. The HylB, HylD, and TolC genes were inserted onto a plasmid designed to express these channel-forming proteins at low levels in the host DH5a E. coli cells. The c-terminal domain of the HylA gene was cloned into a pUC18 plasmid that was engineered with a 6His tag and the Hyl A c-terminus. This vector allowed us to demonstrate the expression and secretion of a green fluorescent protein fusion to HylA, a result that indicates that many different proteins can be secreted by this method. This system should allow for a standardized protocol to be applied to expression and purification of virtually any coding domain, as the expression in our test case did not require a specific induction protocol. In addition, affinity purification was greatly simplified by the fact that the secreted protein made up the majority of protein found in the growth medium. Our goal is to develop a standardized system for E. coli expression-secretion-purification that is applicable to a large number of diverse proteins. Phase I work will include: optimization of the growth/induction protocol, and introduction of a thrombin proteolytic recognition sequence between the HylA and inserted protein sequence; allowing removal of the hylA peptide from the secreted protein. A determination of the minimum portion of HylA that is needed for secretion will be made, and several protein coding regions will be tested with the system; including a toxic protein. Measurements of the enzyme activity of the secreted proteins will be carried out. Project Narrative: The products that we will develop in the form of kits, vectors, and cells lines will be directly marketable to the 40,000 or more molecular biology and proteomics labs through academia, non-profit organizations, biotechnology, and pharmaceutical companies, including the many laboratories supported by the NIH. MacConnell Research Corp. itself can directly market these products, as we have already established a customer base for our existing products. The products resulting from Phase II, if successful, will have a realistic potential of $10-$20 million in sales per year. They will improve protein production and yield high quality protein. These products will be essentially ready for market at the end of Phase II.