A Novel Assay for Measurement of Membrane-Protein Surface Expression using a β-lactamase

    loading  Checking for direct PDF access through Ovid

Abstract

Synopsis

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Here, we describe a simple, low cost, and potentially high-throughput assay, for measurement of surface expression of proteins. The assay uses β-lactamase enzyme (βlac) as a reporter, and its cell-impermeable substrate, nitrocefin. The utility of this assay is demonstrated using well-established paradigms of internalization and molecular chaperoning, applied to two GPCRs and a monoamine transporter.

Synopsis

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one-step and potentially high-throughput assay, using the β-lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell-impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well-established paradigms of internalization and molecular chaperoning, applied to two G-protein-coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression

Synopsis

Symbol

Related Topics

    loading  Loading Related Articles