Immobilized artificial membranes: Their syntheses and applications in membrane protein purification

Songjun Cai, Purdue University

Abstract

Immobilized artificial membranes (IAMs) emulate biological membranes and are currently being exploited to (i) predict the passive transport of drugs through cell membranes and (ii) purify membrane proteins. IAMs are prepared by bonding membrane lipids containing an $\omega$-carboxyl group on the hydrocarbon chain to solid surfaces with the lipid correctly oriented after bonding. IAM surfaces with two acyl chains were prepared by immobilizing analogs of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) onto silica propylamine. PLs that require protection/deprotection during the preparation were enzymatically synthesized using phospholipase A$\sb2$ (PLA$\sb2)$ and phospholipase D (PLD). The IAM surfaces made are designated as $\rm\sp{ester}$IAMs. Single chain ether phospholipids containing $\omega$-carboxyl groups in the alkyl chain were also immobilized to prepare $\rm\sp{ether}$IAMs. Both FTIR spectroscopy and elemental analysis indicated that the bonding densities for the immobilized PLs are very close to the density of phospholipids found in fluid liposome membranes. Studies elucidating the affinity of IAMs for proteins utilized PLA$\sb2$ as a model protein. The elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA$\sb2.$ Elution conditions tested included (i) detergent gradients, (ii) salt gradients, (iii) acetonitrile gradients, and (iv) a two step gradient consisting of a detergent gradient followed by an acetonitrile gradient. PLA$\sb2$ from Sigma was purified to electrophoretic homogeneity with a 417-fold increase in specific activity in one step and PLA$\sb2$ from a freshly prepared mixture was purified in one step with a 660-fold increase in specific activity. A key implication from this work is that IAMs may have particularly good utility in purifying peripheral membrane proteins. Aside from PLA$\sb2,$ N-acylphosphatidylethanolamine synthase (NAPES) was purified using IAMs. NAPES is a membrane bound enzyme present in cotton seedlings at trace concentrations, and it catalyzes acylation of the amino group of membrane PE. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using $\rm\sp{ether}$IAM PEC$\rm\sp{C10/C3}.$ Dimyristoylphosphatidylethanolamine (DMPE), the natural substrate, was required in the mobile phase for elution of optimum functional activity. DMPE functions as both a surfactant-type affinity displacing ligand effecting protein elution and a stabilizing factor of NAPES functional activity during chromatography. This one step IAM purification of NAPES resulted in a 3940-fold increase in NAPES specific activity. The molecular weight of the purified NAPES protein is 64,000. In summary, new IAM surfaces are being synthesized that allow solving several biological problems which include protein purifications.

Degree

Ph.D.

Advisors

Pidgeon, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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