Expression and Purification of Membrane Proteins from Leishmania major for Structural
Genomics. Nadia Fedoriw*, Kathy M. Clark*, Sara M. Connelly*, Katrina Robinson* Earl
Walker*, Ina Urbatsch†, Wim G. Hol‡, and Mark E. Dumont*
*Center for Human Genetics and Molecular Pediatric Disease and Department of Biochemistry and
Biophysics. University of Rochester Medical Center. Rochester, NY 14642.
†
Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, TX
79430-6540
‡
Howard Hughes Medical Institute, Biomolecular Structure Center, University of Washington,
Seattle WA 98195-7742
Predicted transmembrane proteins represent 20-30% of the complement of genes in most
organisms, but there are currently only several dozen known structures of transmembrane proteins,
mostly from prokaryotes. As part of a structural genomics project focusing on pathogenic protozoa,
we are developing a high-throughput system for cloning, expression, and purification of
transmembrane proteins from the parasitic protozoan Leishmania major. We have constructed a
series of vectors that allow the use of ligation-independent cloning for transfer of a single PCRamplified open reading frame into plasmids for expression in E. coli or the methylotropic yeast
Pichia pastoris either with or without yeast or E. coli-specific secretion signal sequences. The
vectors are designed to create C-terminal fusions to 6-His and calmodulin binding peptide affinity
tags that are cleavable by rhinovirus 3C protease. We have amplified and cloned 75 L. major genes
that are predicted to contain two or more transmembrane segments into multiple expression vectors.
We find that 50 of these can be expressed in E. coli at levels sufficient for detection in crude lysates
by immunoblotting and 20 are expressed at levels sufficient for detection in lysates by Coomassie
staining. For almost all of these reading frames, levels of expression in E. coli are greatly enhanced
by addition of a bacterial pelB signal sequence. Although most of the reading frames expressed in
E. coli are found in a low-speed pellet fraction following cell lysis, many of them can be solubilized
by non-ionic detergents, especially members of the fos-choline (acylphosphocholine) series. The
solubilized proteins can be purified by metal ion and calmodulin affinity chromatography and
exchanged into other detergents while bound to affinity columns. In P. pastoris, more than 50% of
the predicted transmembrane reading frames tested for expression can be detected by
immunoblotting in crude lysates at levels corresponding to more than 20 mg/liter of fermentor
cultures. Most of these proteins are present in high-speed pellet fractions indicative of targeting to
membranes. The presence of a yeast secretion signal is does not appear to be critical for expression
of these reading frames in P. pastoris.