GOALI: Efficient Production of Correctly Folded tPA and Other Complex Eucaryotic Proteins in Escherichia coli

9634036乔治欧这项建议正在研究中，以提高在大肠杆菌中正确折叠的复杂真核蛋白的生产。提出以溶栓酶组织纤溶酶原激活物(TPA)为模型蛋白，优化分泌型多二硫键蛋白在细菌中的表达。预计在tPA中最大限度地形成正确的二硫键的条件也将有助于表达具有较少二硫键的更简单的蛋白质。提出了分三步走的战略。这包括：(1)细胞工程：这一方法利用了研究人员最近的一项发现，即哺乳动物蛋白二硫键异构酶(PDI)可以在大肠杆菌中以功能形式表达，在那里它可以调节少量但显著的活性tPA的表达。将确定增强细菌周质空间中PDI活性从而导致正确折叠的tPA产量增加的条件。(2)遗传筛选：将使用遗传筛选来分离产生大量正确折叠tPA的突变体，因为二硫键的形成是一个复杂的过程，单靠细胞工程方法可能很难优化。将分离出具有最高活性tPA滴度的突变体，并对其进行鉴定，以确定导致更有效的二硫键形成的遗传损伤。(3)发酵工程：对已知对tPA折叠有影响的发酵条件的影响进行系统研究。这些参数包括溶解氧浓度、生长介质的氧化还原电位、低分子硫醇和二硫化物的存在以及培养pH。将测定催化二硫键形成的酶的体内氧状态，以便了解生长条件对二硫键机械功能的影响。此外，还将研究在保持异源蛋白质折叠的最佳氧化状态的同时最大化细胞密度的发酵条件。这项研究是与加利福尼亚州旧金山南部的基因技术公司合作进行的。为了完成这项研究，基因泰克将投入大量资源，包括科学家的时间发酵设备、表达载体、抗体、分析支持、tPA样本等。***

9634036 Georgiou This proposal is on research to improve the production of correctly folded complex eucaryotic protein in E. coli. It is proposed to employ thrombolytic enzyme tissue plasminogen activator (tPA) as the model protein for optimizing the expression of secreted multidisulfide proteins in bacteria. It is expected that conditions which maximize the formation of the correct disulfide bonds in tPA will also be useful for the expression of simpler proteins with fewer disulfide bonds. A three part strategy is proposed. This includes: (1) Cellular Engineering: This approach utilizes a recent discovery by the investigators that the mammalian protein disulfide isomerase (PDI) can be expressed in a functional form in E. coli where it can mediate the expression of low, but significant, amounts of active tPA. Conditions that enhance the activity of PDI in the bacterial periplasmic space, and thus result in increased yields of correctly folded tPA, will be determined. (2) Genetic Screening: The use of genetic screens to isolate mutants producing high amounts of correctly folded tPA will be employed since disulfide bond formation is a complex process that may prove difficult to optimize solely by cell engineering approaches. Mutants exhibiting the highest titer of active tPA will be isolated and characterized to determine the genetic lesions that result in more efficient disulfide bond formation. (3) Fermentation Engineering: A systematic study of the effect of fermentation conditions which are known to have an influence on tPA folding will be conducted. These include parameters such as dissolved oxygen concentration, the redox potential of the growth medium, the presence of low molecular weight thiols and disulfides and culture pH. The in vivo oxygen states of the enzymes that catalyze disulfide bond formation will be determined so that the effect of growth conditions on function of the disulfide machinery can be understood. Also, fermentation c onditions that maximize cell density while maintaining the optimal oxidation state for heterologous protein folding will be investigated. This research is a cooperative activity with Genentech Inc. in South San Francisco, California. Genentech will commit significant resources including scientists' time fermentation equipment, expression vectors, antibodies, analytical support, tPA samples etc. towards the completion of this research. ***