Biomedical Engineering Reference
In-Depth Information
Chapter 8
A
β
-Lactamase Based Assay to Measure Surface
Expression of Membrane Proteins
Pieter Beerepoot , Vincent M. Lam , and Ali Salahpour
Abstract
Measurement of cell surface expression is an essential part of studying membrane proteins. Traditional
techniques for measuring surface expression depend on the availability of appropriate radioligands or anti-
bodies towards extracellular epitopes of a protein of interest. The current protocol outlines the use of an
assay to monitor surface expression of membrane proteins tagged with a bacterial
ʲ
-lactamase in mammalian
cell lines. The use of this technique allows for quick, quantitative, sensitive, and inexpensive measurement
of surface expression, with the potential for high-throughput screening.
Key words
Surface expression, Membrane proteins, Beta-lactamase, GPCR, Transporter
1
Introduction
The functions of membrane proteins at the cell surface, particularly
receptors and transporters, are tightly regulated and dynamic due
to internalization and recycling processes. Radioligand binding,
surface biotinylation, and antibody-dependent assays, including
fl ow cytometry or enzyme-linked immunosorbent assays (ELISA)
have been traditionally used to quantify surface expression of mem-
brane proteins [
1
]. These assays are time-consuming and require
reagents that are expensive or often may not be available for a par-
ticular protein of interest. We recently developed a new compli-
mentary method for measuring surface expression. In our assay, we
fused
-lac) to extracellular motifs of the proteins of
interest and quantifi ed surface expression by measuring
ʲ
-lactamase (
ʲ
ʲ
-lac activ-
ity using a cell impermeable substrate.
ʲ
-lac is a bacterial enzyme that hydrolyzes the
ʲ
-lactam ring
present in penicillins.
-lac has previously been used in various
reporter assays [
2
-
5
]. A number of substrates for this enzyme are
available, which either produce a colorimetric change, or a shift in
fl uorescence properties upon hydrolysis of the
ʲ
-lactam ring [
2
,
5
].
In our surface expression assay, the colorimetric substrate nitrocefi n
ʲ
