Immobilized artificial membrane chromatography for the purification of membrane proteins
Abstract
A novel chromatographic support denoted as an immobilized artificial membrane (IAM) has been designed and developed specifically for the purification of membrane proteins. The main goal of IAM solid supports is to provide a lipid environment similar to cell membranes. The membrane-like IAMs are prepared by covalently linking analogs of cell membrane phospholipids on to solid surfaces. The IAM bonded phases that have been prepared to date include $\sp{\rm ether}$IAM.PC$\sp{\rm C10/C3}$, $\sp{\rm ether}$IAM.PG$\sp{\rm C10/C3}$, $\sp{\rm ether}$IAM.PE$\sp{\rm C10/C3}$, $\sp{\rm ether}$IAM.PS$\sp{\rm C10/C3}$, and $\sp{\rm ether}$IAM.PA$\sp{\rm C10/C3}$. The superscript ether denotes that a long alkyl chain is linked to the glycerol backbone by an ether bond. The superscript C10/C3 denotes that $\sp{\rm ether}$IAM surfaces are endcapped with decanoic (C10) and propionic (C3) anhydride, respectively. Both Fourier transform infrared studies and elemental analysis indicated that the bonding densities are 64-83 mg of phospholipids/g of silica propylamine, which corresponds to a monolayer density. In addition, $\sp{\rm ether}$IAM.PC$\sp{\rm C10/C3}$ supports have been evaluated as high performance liquid chromatographic column using amphetamine analogs under isocratic conditions at different pHs from pH 2 to pH 8.5. After perfusing 25 liters (50,000 column volumes) of 0.1 M ammonium phosphate buffer, $\sp{\rm ether}$IAM.PC$\sp{\rm C10/C3}$ HPLC columns show stability at acidic pH but not at basic pH. By utilizing IAM HPLC, the human intestinal peptide transport (HPT-1) protein was partially purified from human intestinal Caco-2 cells. The HPT-1 protein has an apparent mass of $\sim$120 kD and was partially purified using an $\sp{\rm ether}$IAM.PG$\sp{\rm C10/C3}$ HPLC column with an 1% CHAPS detergent gradient. The pooled fractions that contained highly enriched HPT-1 protein were further purified by gel filtration chromatography. Immobilized artificial membranes are a very efficient preliminary step for the purification of the HPT-1 protein.
Degree
Ph.D.
Advisors
Pidgeon, Purdue University.
Subject Area
Analytical chemistry|Biochemistry|Organic chemistry
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