Immobilized Artificial Membrane (IAM) technology involves covalently bonding membrane lipids to solid surfaces through the lipid alkyl chains. For Phase II of the project, the applicants will expand the selectivity of IAM surfaces by preparing at least 9 different IAMs. The new IAM surfaces will be derived from pure or mixed lipid systems using phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid. A KIT will be assembled which contains all the IAMS prepacked in cartridges for quick-and-easy affinity studies. The KIT will contain 50 mg-IAM/cartridge, and the purpose of the KIT is to simultaneously characterize the binding properties of many IAMs for many membrane proteins in one experiment. Loading the solubilized sample on each IAM cartridge in the KIT, followed by gel electrophoresis of the filtrate, provides a means to rapidly identify which proteins do not bind to the IAM surface, i.e., the affinity of the IAM for the target protein can be rapidly evaluated using the KIT. Each filtrate from the 9 IAM cartridges is expected to have a different composition of non adsorbed proteins. Thus the IAM KIT will rapidly identify which bonded phases have affinity for any given membrane protein. The IAM KIT will be evaluated using crude membrane preparations from (1) Red cell membranes, (2), E-Coli expression systems, (3) Squid brain extracts, and (4) Human T-cells. Several laboratories are participating in the evaluation of the IAM KIT and these labs will supply solubilized samples, methods for analysis of functional proteins, and they will also perform some of the experiments in their own lab. The IAM KIT should be able to identify the correct surface for purifying any membrane protein from any solubilized sample and the appropriate IAM column can then be purchased with confidence that it will effect the desired separation. The IAM column packing material synthesized during Phase II of the SBIR project will contain ether-lipids to assure high product quality as was demonstrated during Phase I.
