Core B, the Protein Expression and Production Core, will express and purify integral membrane protein transporters as required for each individual project. In some cases expression and purification methods have already been developed, and the Core then provides supply to the projects as required in timely fashion. In other cases particularly when screening homologs and truncations, or mutated versions of a gene procedures require a new strategy for optimization of expression, extraction and purification procedures. One goal is to supply pure, homogenous, ABC transporters, and in the form most suited to use by; Project 1 ?Conformational Fabs for dissecting transporter structure and function? for Fab selection against different conformational states; to Project 2 Mapping the Conformational Cycle of Transmembrane Transporters to support cryo-EM imaging; and to Project 3 ?Structure, States and Transitions in ABC Transporters for crystallography and other structural methods. MRP4, a eukaryotic target ABC transporter has been expressed and purified. It can be supplied in required amounts to Projects 1,2,3; however optimizing expression is a major focus of Core B. Other species and constructs of TAPL, and eukaryotic multi drug transporters will be designed and produced as needed. Other proteins mentioned include TAP1/2 species, where core B will attempt improved expression, and human AQP4 where robust production will be supplied as needed, to project 3. Genes will be redesigned according to algorithms we helped to develop, synthesized and expressed in multiple expression systems available in the Stroud lab. The pathway for eukaryotic membrane proteins begins with trials in the Saccharomyces system that we have developed, and progresses to transient expression in HEK cells (2 days), viral transformation of insect cells (3 weeks) and stable gene integration into HEK GnTI- cells grown in suspension (3 months). Fluorescent proteins fused to target proteins facilitate analysis of levels of expression, their location inside the cell, and quality of expression. These probes also allow initial testing of panels of detergents and initial optimization of buffer conditions in crude extracts for those that best maintain the sample in solution. Target proteins are engineered with cleavable affinity tags for purification. Characterization includes Tetradetector analysis of size exclusion chromatography. This scheme allows for clear separation and removal of excess detergent-lipid micelles from the protein containing micelles of purified protein-detergent-lipid complexes. Mass spectrometry is used to characterize the mass of pure membrane proteins, to identify any endogenous lipids including cholesterol that are bound, or required by the protein, and to identify post- translational processing that may affect homogeneity.
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