Beyond bottlenecks in membrane protein production - III
Erythromycin resistance protein as a expression reporter
Daniel Linares, Eric Geertsma and Bert Poolman
Department of Biochemistry, University of Groningen, Nijenborgh 4, Groningen 9747 AG, NL
Phone: +31 50 3634208; FAX: +31 50 3634165; e-mail: d.martinez.linares@rug.nl
Integral membrane proteins (IMP) represent the most challenging macromolecules in the field of
structural genomics. The overproduction of biologically active material is a major bottleneck in
the functional and structural characterisation of IMPs. Recent advances in the development of
Lactococcus lactis as high-throughput expression system have increased the potential to
heterologously produce prokaryotic and eukaryotic membrane proteins and expanded the
expression space (1). Unfortunately, not every protein is functionally overexpressed in L. lactis or
E.coli. The use of efficient screening and selection methods, based on fusion reporters, to
optimize growth conditions and host strains would tremendously facilitate the exploitation of
existing expression systems (2). It would allow one to develop strains furnished with an increased
capacity to produce well-folded proteins in high amounts.
Here, we show that the erythromycin resistance protein (ERY), when fused to an IMP, can be
used as an indicator to monitor the level of functional protein in L. lactis. Survival of L. lactis
cells on media containing erythromycin correlated positively with the level of correctly folded
protein. Furthermore, overexpressed IMP could be recovered from IMP-ERY fusions, using a
site-specific protease. Taken together, these properties make ERY an excellent tool for largescale IMP target selection in structural genomics projects. At the meeting, we will present a
comparison of ERY and GFP as fusion partners for screening of functional overexpression.
1. Geertsma, E.R., and Poolman, B. (2007) High-throughput cloning and expression in
recalcitrant bacteria. Nature Meth. 4, 705-707.
2. Geertsma, E.R., Groeneveld, M., Slotboom, D.J., and Poolman, B. (2008) Quality control of
integral membrane proteins. Proc. Natl. Acad. Sci. USA. 105, 5722-5727.