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

University Of Pittsburgh, Pittsburgh PA

Investigators

Abstract

Ataai 0533949 This 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 laboratory conditions, (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.