1. PURIFICATION OF YEAST MEMBRANE PROTEINS FOR STRUCTURAL GENOMICS Center for High Throughput Structural Biology Mark E. Dumont *†, Nadia Fedoriw *, Kathy Clark *, Katrina Robinson *, Gayle Schneider * Michael G. Malkowski ‡, George T. DeTitta ‡, and Mark Sullivan * * 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 Conclusions 1. About 250 yeast transmembrane proteins can be overexpressed to levels of ~ 1mg per liter of culture. The best yields of purified protein are ~0.3 mg/l. 2. Detergents of the fos-choline and maltoside families are generally effective for initial solubilization of many yeast membrane proteins. 3. Efficiency of cleavage of tags on membrane proteins by rhinovirus 3C is much less than for soluble proteins protease and is variable, depending on the target. However intrinsic 3C activity is not inhibited by detergent. 4. IgG binding of ZZ domain tags provides effective purification from whole-cell lysates. Immobilized metal affinity purification is much less efficient. 5. High-purity yeast transmembrane transmembrane proteins are now being produced for crystallization and have successfully served as antigens for generating recombinant single chain antibodies for co-crystallization. Summary As part of the Center for High-Throughput Structural Biology we are developing technologies for efficient use of yeast for expression and purification of endogenous yeast transmembrane proteins (TMPs) for x-ray crystallography. This combines the benefits of homologous expression of proteins with the advantages of yeast as an organism with well- developed and tractable genetics that is inexpensive to culture. Furthermore, yeast are the only organisms that have been successfully used for heterologous expression of eukaryotic membrane proteins for x-ray crystallography. 1,2 We have focused initially on a set of endogenous TMPs that are the highest expressing reading frames in a previously-constructed genomic collection of Saccharomyces cerevisiae expression clones. 3 High-expressing TMP targets have been selected based on the availability of established biochemical assays for determining the whether the protein is maintained in a native state. Ligation independent cloning and Gateway  cloning procedures have been used to transfer reading frames into vectors that allow galactose-controlled expression of reading frames containing cleavable C- terminal affinity tags. Efficient purification of the solubilized yeast-expressed proteins has been achieved based on affinity chromatography using immobilized metal and IgG affinity matrices with elution by imidazole and by cleavage of tags using rhinovirus 3C protease. However, 3C protease cleavage of target TMPs has been found to be variable and, generally, less efficient than for soluble proteins with similar tags. Yields of purified protein are generally less than 0.3 mg per liter of high-density fermentor-grown culture. Purified proteins and proteins in cell membranes are being used to generate recombinant single chain antibodies to aid in crystallization. 1 Long SB, Campbell EB, and Mackinnon R.(2005) Science. 309, 897-903. 2 Jidenko M, Nielsen RC, Sorensen TL, Moller JV, le Maire M, Nissen P, Jaxel C. (2005) Proc Natl Acad Sci U S A. 102, 11687-91. 3 Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, and Grayhack EJ. (2005) Genes Dev. 19, 2816-2826. 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. 3 (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 two structures of heterologously expressed eukaryotic transmembrane proteins have been solved by x-ray crystallography. 1,2 Both of these were based on proteins 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 pSGP38 (Ligation independent cloning) P GAL PGK1 5’ LIC site ORF3CHis6ZZ P GAL ORF3CHis6ATT site HAZZ Membrane Protein Expression Levels in Yeast 64 - 48 - 37 - 26 - 19 - 15 - 6 - 82 - 115 - 180 - IgG LC IgG HC Yeast cultures were grown to 15 OD 600 then lysed by vortexing with glass beads. Lysates were solubilized in 0.1% Fos-choline 16, bound to IgG sepharose, washed, then eluted in SDS loading buffer containing 9 M urea. Each lane contains the equivalent of 10 mls of the original culture.The indicated molecular weights refer to the un-tagged ORFs. (Tag molecular weight is 18kDa.). MarkerIgG alone AAC1 (34 kD)AAC3 (33 kD)CHO1 (31 kD) YNL275W (65 kD) PMP3 (6 kD)RCE1 (36 kD)RHK1 (53 kD) PMT5 (85 kD) 3C Protease Cleavage of Target from IgG-Sepharose MarkerElution 1Elution 2Stripped IgG 1/5 X 18 uL IgG AloneElution 1Elution 2Stripped IgG 1/3 X Elution 1Elution 2Stripped IgG 1/2 X Elution 1Elution 2Stripped IgG 1 X Elution 1Elution 2Stripped IgG 2 X Relative 3C-GST protease added IgG LC IgG HC 3C-GST Cleaved YNL275w Uncleaved YNL275w Cleaved tag 21 ug 3C-GST Cultures of a yeast strain expressing YNL275w (putative yeast borate transporter related to mammalian anion transporters) in the MORF library vector were lysed, solubilized in 1% dodecylmaltoside (DDM), then bound to IgG-Sepharose. The bound material was washed in 0.01% DDM, then subjected to cleavage in the presence of various amounts of GST-tagged rhinovirus 3C protease (“Elution” lanes.) Protein remaining on the IgG-Sepharose after cleavage (including IgG) was then eluted in SDS-containing gel- loading buffer (“Stripped.IgG” lanes). Each lane was loaded with the equivalent of 250 OD  ml of culture. Advantages of Cleavage of IMAC Affinity Tags Imidazole Elutions AAC1p 3C Cleavage Marker Elution 1Elution 2Elution 3Stripped Talon 4  g 3C-His6 AAC1p Yeast cells expressing AAC1 (adenine nucleotide transporter) were lysed by vortexing with glass beads, solubilized in 0.5% fos-choline 16, bound to Talon  immobilized metal affinity matrix, then washed with buffer containing 0.1% dodecylmaltoside. Elutions were performed by treatment with the indicated concentrations of imidazole (left panel) or by overnight incubation at 4 o C with His6-tagged 3C protease (right panel). Each lane was loaded with material derived from 200 OD  ml of culture. 182 kD - 115 kD - 82 kD - 64 kD - 49 kD - 37 kD - 26 kD - 19 kD - 15 kD - 6 kD - Marker 5 mM 15 mM50 mM 150 mM 300 mM 500 mM-1 0.1 M EDTA +SDS 500 mM-2 AAC1p + tag Gel Filtration of Affinity Purified Anion Transporter 171 109 79 60 47 35 25 18 14 6 Marker IgG Purif. 1 st Gel Filt. 58 59 60 61 62 63 64 65 66 67 68 69 70 Fractions YNL275W 3C-GST 71 72 73 74 75 76 77 78 79 Fractions Absorbance (280nm) Fractions Cells expressing the putative anion tranporter encoded by YNL275w from the MORF vector were lysed using an Avestin high pressure microfluidic device, solubilized in 1% dodecylmaltoside, bound to IgG-Sepharose, washed with buffer containing 0.01% dodecylmaltoside, and eluted by overnight incubation at 4 o C with GST-tagged 3C protease. The eluate was then subjected to two rounds of gel filtration on Superdex 200.