1. Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology Kathy Clark *, Nadia Fedoriw *, Katrina Robinson *, Mark Sullivan †, Michael G. Malkowski ‡, George T. DeTitta ‡, and Mark E. Dumont †* * Department of Pediatrics and † Department of Biochemistry and Biophysics University of Rochester Medical Center Rochester, NY 14642 and ‡ The Hauptman-Woodward Institute, 700 Ellicott Street, Buffalo, New York 14203 Current bottlenecks/solutions 1. High-purity yeast transmembrane proteins are now being produced for crystallization and have successfully served as antigens for generating recombinant single chain antibodies for co- crystallization. The best yields of purified protein are 0.3 mg/l of culture. 2. The goal of “E. coli-fying” yeast as an expression system for membrane proteins will benefit from ongoing development of improvements in the following areas: - Development of culture and induction conditions leading to increased overall expression of folded proteins. - Use of repeated cycles of cell lysis for more complete recovery of targets. - Selection of optimum detergent for efficient solubilization based on recent genome-scale surveys of detergent effectiveness such as that of White et al. (2007). - Development of purification protocols that do not rely on cleavage of tags or engineering of specific proteases with enhanced activity toward detergent-solubilized proteins. - Development of rapid purification protocols that maintain a population of protein-bound lipids. - Maintenance of high protein concentration throughout purification to avoid extensive concentration of detergent in final steps. Summary To address the severe lack of three dimensional structural information for eukaryotic transmembrane proteins (TMPs), the Center for High-Throughput Structural Biology is developing protocols for expression and purification of TMPs in the yeast Saccharomyces cerevisiae. We have focused initially on a set of endogenous yeast TMPs that are the highest expressing reading frames in a previously-constructed genomic collection of S. cerevisiae expression clones and for which there are established biochemical assays for determining whether the protein is maintained in a native state. Genes encoding the target TMPs are transferred via ligation-independent cloning procedures to a series of vectors that allow galactose-controlled expression of reading frames fused to C- terminal His6, His10, and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Single chain antibodies (scFvs) recognizing several targets have been developed as aids to crystallization and purification. Current efforts are focused on overcoming bottlenecks in protein production and crystallization by introducing the following improvements at different levels of the production pipeline: 1) improving overall levels of cellular expression of TMPs by altering protocols for cell growth and induction of expression; 2) increasing efficiency of cell lysis; 3) increasing the efficiency of detergent solubilization; 4) increasing the yield of 3C protease cleavage; 5) reducing the number of steps required for effective purification; 6) optimizing the amount of residual lipid purifying with the TMP; 7) developing protocols that allow production of highly concentrated protein solutions that do not also contain high detergent concentrations; 8) the use of additives such as lipids and enzyme inhibitors to stabilize purified proteins. Targeting Strategies 30 Target ORFs are currently selected based on the following criteria: 1. Prediction of two or more transmembrane segments based on TMHMM and HMMTop 2. Absence of evidence that ORF is part of a hetero-multimeric complex, based on genomic/proteomic databases. 3. High level expression in C-terminal-tagged genomic Saccharomyces cerevisiae MORF library of Gelperin et al. (2005). (263 predicted integral membrane proteins in MORF library are expressed at levels of ~1mg/l. Of these, 90 have human orthologs) 4. Existence of a published procedure for assaying native state of produced protein. Yeast Membrane Proteins Expressed in Yeast 1. To date, only three structures of heterologously expressed eukaryotic transmembrane proteins have been solved by x-ray crystallography. Both of these proteins were expressed in yeast. 2. Advantages of homologous expression system for post-translational modifications, membrane targeting, protein folding, lipid requirements 3. Extensive annotation of yeast genome as far as protein-protein interactions, subcellular localization, expression levels, protein function 4. Availability of yeast strains with altered protein degradation, unfolded protein response, post- translational modifications, intracellular trafficking 5. Rapid and inexpensive conditions for culturing yeast cells Vectors for yeast membrane protein expression MORF library vector (Gateway cloning) 1 pSGP36 (Ligation independent cloning) P GAL PGK1 5’ LIC site ORF3CHis10 pSGP40 (Ligation independent cloning) P GAL PGK1 5’ LIC site ORF3CHis10ZZ P GAL ORF3CHis6ATT site HAZZ Fermentor culture (autoinduction galactose) Lysate Sup Bind to IMAC or IgG affinity matrices Harvest, lyse (Avestin) 100,000 x g spin Gel filtration Concentrate Pellet 3,000 x g spin SupMembrane Pellet 1.2M KCl; 120,000 x g spin Detergent solubilization 26,000 x g spin Pellet (solubilized protein) Static Light Scattering Crystallization trials ORF cloning Target selection Salt-washed membranes 3C protease cleavage Imidazole elution Detergent exchange and dilution Culture conditions: Issues S. cerevisiae achieves >100 g/liter (dry cell weight) in fermentation on rich media BUT: Plasmid losses of ~50% are observed for some of our strains on rich medium ALSO: We find that growth at low temperatures (26 o C) stabilizes some membrane proteins against subsequent precipitation. Talon-binding proteases of yeast Ste24p cleaved from Talon with GST-tagged 3C protease Ste24p stripped from Talon using EDTA Strain 1: BJ5460 pep4 - prb1  Strain 2: EJG1117 pep4 - prb1 - Strain 3: EJG1364 pep4 - PRB1 + Each purification: 300 OD mls Ste24-40 uncleaved Ste24-40 cleaved 3C-GST PMSF + - + - + - + - + - + - 123123 Strain Endogenous yeast proteases that degrade the Ste24p target as well as 3C protease include protease B (Prb1p) and can be inhibited by PMSF (but not all serine protease inhibitors.) Markers 0.1 M NH 4 Br 0.1M Tris pH 8 20% PEG 8000 0.1 M NH 4 Br 0.1M Acetate pH 5 20% PEG 8000 0.2 M KSCN pH 7 20% PEG 3350 Anion transporter YNL275w (pSGP40, cleaved) KCl-stripping of membranes 275W-40 from IgGMarker, 15 uLWash 1Wash 2 5 mM imidazole 15 mM imidazole 50 mM imidazole 150 mM imidazole300 mM imidazole 500 mM imidazole EDTA-Stripped Amount of YNL275W-40 1/6 liter Loading: 1/200 th ofpurification 275W-40, from IgG Marker5 mM imidazole15 mM imidazole 50 mM imidazole 150 mM imidazole300 mM imidazole 500 mM imidazole EDTA-Stripped YNL275w-40 Un-stripped membranes0.7 M KCl-stripped membranes Marker 50 kDa concentrate 50 kDa filtrate 100 kDa concentrate 100 kDa filtrate Concentration of purified protein in the presence of detergent Comparison of 50 kD-cutoff (expected to retain DDM micelles) and 100 kD-cutoff (expected to pass DDM micelles 1 ) membranes in purification of Ste24p (CAAX protease.) Marker 3C-His6 elution 500 mM imidazole elution 20 ul 10 ul 5 ul 3C-6HIS protease ( 7 ug) 3C-GST protease ( 5 ug) 49 kDa Purification from 96,000 OD mls 1-Step Purification of Ste24p (CAAX protease) on Talon  Ste24p expressed from vector pSGP40 was solubilized from KCl- washed membranes, bound to Talon, then eluted by cleavage with His6- tagged 3C protease. After elution, the Talon column was treated ith 500 mM imidazole to visualize Fraction # Gel Filtration Superdex 200 Ynl275w Multi-step purification of the anion transporter YNL275w Talon Elution 1 Talon Elution 2 Talon Elution 3 Urea/SDS stripped Talon IgG super rebound to IgG IgG Elution 1 IgG Elution 2 IgG Elution 3 IgG stripped urea/SDS Elutions after GST resin Ynl275w (cleaved) 3C-GST Ynl275w (un-cleaved) Marker Yln275w 10  l Ynl275w 5  l 3C-GST 5  g Detergent: dodecyl maltoside Culture: 96,000 OD  mls The C-terminal tags of many yeast membrane proteins may be obscured by detergents 1. Many tagged yeast membrane proteins are not efficiently cleaved by 3C protease 2. The activity of 3C protease is not intrinsically sensitive to detergents. 3. Inefficient cleavage can sometimes be overcome by adding large amounts of protease. 4. Affinity tags on yeast membrane proteins do not appear to be as accessible as the same tags on soluble proteins (His10 is useful but His6 generally is not.) 5. Also: Use of Nickel-NTA resin inhibits subsequent 3C protease cleavage whereas use of cobalt (Talon  ) does not. tag (Z-domain) protein detergent 3C-cleavage site His-His-His-His-His-His