Collaborative Research: Efficient Bioseparation by Intertwining Strain, Chromatography, and Affinity Tail Design

Beitle0534836该提案是与匹兹堡大学（BES 0533949）的合作研究，其描述了通过固定化金属亲和色谱（IMAC）技术的新的蛋白质优化纯化方法。 如果成功，这项工作将减少由于污染蛋白质吸附而造成的柱容量损失，并降低蛋白质洗脱方案的复杂性。 这项工作包括使用pH和交替咪唑梯度来定义大肠杆菌蛋白质的IMAC洗脱曲线的低背景区域，使用二维凝胶电泳、质谱和数据库分析的组合来鉴定这些蛋白质，如果在实验室条件下不需要生长，则删除编码蛋白质的基因，（或通过特异性突变改变它们，使得它们不再在宿主洗脱谱的低蛋白背景区中重叠），并开发高效的金属结合肽以引导洗脱至低背景区域。用于开发IMAC改进的方法广泛适用于使用亲和尾的所有亲和系统。因此，所提出的工作将催化生物分离的一般改进策略。 此外，从事该项目的学生将在生物化学工程和分子生物学的工具方面得到广泛的培训，从而为一代能够领导生物技术的跨学科工程师和科学家做出贡献。

Beitle0534836This proposal is a collaborative study with the University of Pittsburgh (BES 0533949) which describes a new protein optimization purification methodology via the technique of immobilized metal affinity chromatography (IMAC). If successful, the work will reduce columncapacity loss due to contaminating protein adsorption, and reduce the complexity of protein elution protocols. The work involves defining the low background region of the IMAC elution profile of E.coli proteins using both pH and alternately imidazole gradients, identifying these proteins using a combination of 2D gel electrophoresis, mass spectrometry, and database analysis, deleting the genes encoding proteins if they are unnecessary for growth under laboratoryconditions, (or altering them via specific mutation such that they no longer elute in the low protein background region of the host elution profile), and developing highly efficient metal binding peptides to direct elution towards the low background region.The methodology used to develop improvements for IMAC is broadly applicable to all affinity systems employing affinity tails. Therefore, the proposed work will catalyze a general improvement strategy for bioseparation. In addition, the students working on this project will become broadly trained in the tools of biochemical engineering as well as molecular biology thus contributing to a generation of interdisciplinary engineers and scientists capable of biotechnology leadership.