Expression and Purification of Integral Membrane Proteins from
Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology Kathy Nadia Katrina Mark ‡ ‡ †* Michael G. Malkowski , George T. De. Titta , 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 p. SGP 40 (Ligation independent cloning) ZZ His 10 Fermentor culture (autoinduction galactose) Culture conditions: Issues 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. 3, 000 x g spin Lysate Each purification: 300 OD mls - Markers (solubilized protein) Pellet 1 -Step Purification of Ste 24 p (CAAX protease) on Talon 49 k. Da Bind to IMAC or Ig. G affinity matrices Detergent exchange and dilution His-His-His-His tag (Z-domain) 3 C-cleavage site 5. Also: Use of Nickel-NTA resin inhibits subsequent 3 C protease cleavage whereas use of cobalt (Talon ) does not. 3 C protease cleavage Imidazole elution Anion transporter YNL 275 w (p. SGP 40, cleaved) 0. 1 M NH 4 Br 0. 1 M Tris p. H 8 20% PEG 8000 0. 1 M NH 4 Br 0. 1 M Acetate p. H 5 20% PEG 8000 Gel filtration Concentrate Static Light Scattering Crystallization trials 100 k. Da filtrate 0. 2 M KSCN p. H 7 20% PEG 3350 Ste 24 p expressed from vector p. SGP 40 was solubilized from KClwashed membranes, bound to Talon, then eluted by cleavage with His 6 tagged 3 C protease. After elution, the Talon column was treated ith 500 m. M imidazole to visualize Purification from 96, 000 OD mls Concentration of purified protein in the presence of detergent 100 k. Da concentrate 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 cocrystallization. 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. Detergent solubilization 26, 000 x g spin 50 k. Da filtrate Current bottlenecks/solutions Sup Salt-washed membranes 50 k. Da concentrate 3 C-GST 5 g detergent 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 (His 10 is useful but His 6 generally is not. ) 1. 2 M KCl; 120, 000 x g spin protein 2. The activity of 3 C protease is not intrinsically sensitive to detergents. EDTA-Stripped Ste 24 p stripped from Talon using EDTA 500 m. M imidazole Ste 24 p cleaved from Talon with GST-tagged 3 C protease Membrane Pellet 300 m. M imidazole Endogenous yeast proteases that degrade the Ste 24 p target as well as 3 C protease include protease B (Prb 1 p) and can be inhibited by PMSF (but not all serine protease inhibitors. ) Sup 150 m. M imidazole 3 C-GST 50 m. M imidazole Strain 1: BJ 5460 pep 4 - prb 1 Strain 2: EJG 1117 pep 4 - prb 1 Strain 3: EJG 1364 pep 4 - PRB 1+ 15 m. M imidazole 100, 000 x g spin Ste 24 -40 uncleaved Ste 24 -40 cleaved Pellet 500 m. M imidazole elution + - + 3 Loading: 1/200 th ofpurification 3 C-His 6 elution 2 Amount of YNL 275 W-40 1/6 liter 3 C-His 6 elution 1 YNL 275 w-40 5 m. M imidazole Ynl 275 w 5 l Harvest, lyse (Avestin) Marker 1 2 3 Strain PMSF + - + - 0. 7 M KCl-stripped membranes 275 W-40, from Ig. G Yln 275 w 10 l Un-stripped membranes Marker KCl-stripping of membranes S. cerevisiae achieves >100 g/liter (dry cell weight) in fermentation on rich media 3 C-GST protease ( 5 ug) LIC site 3 C 3 C-6 HIS protease ( 7 ug) ORF EDTA-Stripped Elutions after GST resin PGK 1 5’ LIC site 5 ul PGAL 4. Existence of a published procedure for assaying native state of produced protein. 1. Many tagged yeast membrane proteins are not efficiently cleaved by 3 C protease Ynl 275 w 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 ~1 mg/l. Of these, 90 have human orthologs) LIC site 3 C His 10 500 m. M imidazole Ig. G stripped urea/SDS ORF 300 m. M imidazole Ig. G Elution 3 PGK 1 5’ LIC site 150 m. M imidazole Ig. G Elution 2 ORF cloning p. SGP 36 (Ligation independent cloning) PGAL 2. Absence of evidence that ORF is part of a hetero-multimeric complex, based on genomic/proteomic databases. 50 m. M imidazole Ig. G Elution 1 1. Prediction of two or more transmembrane segments based on TMHMM and HMMTop ZZ 15 m. M imidazole Ig. G super rebound to Ig. G 3 C 5 m. M imidazole Urea/SDS stripped Talon HA Wash 2 Talon Elution 3 His 6 Wash 1 Talon Elution 2 ATT site Marker, 15 u. L Talon Elution 1 ORF The C-terminal tags of many yeast membrane proteins may be obscured by detergents Fraction # Gel Filtration Superdex 200 ATT site 10 ul PGAL 275 W-40 from Ig. G Detergent: dodecyl maltoside Culture: 96, 000 OD mls Targeting Strategies 30 Target ORFs are currently selected based on the following criteria: Talon-binding proteases of yeast Multi-step purification of the anion transporter YNL 275 w 3 C-GST Target selection MORF library vector (Gateway cloning)1 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, posttranslational modifications, intracellular trafficking 5. Rapid and inexpensive conditions for culturing yeast cells † Sullivan , Marker 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 Cterminal His 6, His 10, and ZZ (Ig. G-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3 C 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 (sc. Fvs) 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 3 C 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. Ynl 275 w (cleaved) * Robinson , Vectors for yeast membrane protein expression Summary Ynl 275 w (un-cleaved) * Fedoriw , 20 ul * Clark , Comparison of 50 k. D-cutoff (expected to retain DDM micelles) and 100 k. D-cutoff (expected to pass DDM micelles 1) membranes in purification of Ste 24 p (CAAX protease. )
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