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

9634036 Georgiou该提案是关于提高大肠杆菌中正确折叠的复合真核蛋白的生产的研究。 杆菌 建议采用溶栓酶组织纤溶酶原激活剂（tPA）作为模型蛋白，用于优化分泌的多二硫键蛋白在细菌中的表达。 预期使tPA中正确二硫键形成最大化的条件也可用于表达具有较少二硫键的较简单蛋白质。 提出了三个部分的战略。 这包括：（1）细胞工程：这种方法利用了研究人员最近的发现，即哺乳动物蛋白质二硫键异构酶（PDI）可以在大肠杆菌中以功能形式表达。大肠杆菌中，它可以介导少量但大量的活性tPA的表达。 将确定增强细菌周质空间中的PDI活性并因此导致正确折叠的tPA的产率增加的条件。(2)基因筛查：将使用遗传筛选来分离产生大量正确折叠的tPA的突变体，因为二硫键形成是一个复杂的过程，可能难以仅通过细胞工程方法进行优化。 将分离并表征表现出活性tPA的最高滴度的突变体，以确定导致更有效的二硫键形成的遗传病变。 (3)发酵工程：将对已知影响tPA折叠的发酵条件的影响进行系统研究。 这些包括参数，如溶解氧浓度，生长培养基的氧化还原电位，低分子量硫醇和二硫化物的存在和培养pH值。将确定催化二硫键形成的酶的体内氧状态，以便可以理解生长条件对二硫键机制功能的影响。 此外，发酵条件下，最大限度地提高细胞密度，同时保持最佳的氧化状态异源蛋白质折叠将进行调查。 本研究是与Genentech Inc.的合作活动。在南旧金山弗朗西斯科，加州。 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样品等，以完成这项研究。 ***

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. ***