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

这项建议是与阿肯色大学(BES 0534836)的合作研究，该研究描述了一种通过固定化金属亲和层析(IMAC)技术优化蛋白质纯化的新方法。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和交替的咪唑梯度来确定大肠杆菌蛋白的iMac洗脱图谱的低本底区域，使用2D凝胶电泳、质谱学和数据库分析的组合来鉴定这些蛋白质，如果编码蛋白质的基因对实验室条件下的生长是不必要的，则删除它们(或通过特定的突变改变它们，使它们不再在宿主洗脱图谱的低蛋白背景区域洗脱)，以及开发高效的金属结合肽以引导洗脱朝向低本底区域洗脱。因此，拟议的工作将促进生物分离的一般改进战略。此外，参与该项目的学生将在生物化学工程和分子生物学工具方面得到广泛的培训，从而为培养能够领导生物技术的新一代跨学科工程师和科学家做出贡献。

Ataai0533949This proposal is a collaborative study with the University of Arkansas (BES 0534836) 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.