Journal of Structural Biology Hebrew University of Jerusalem

Journal Of Structural Biology Hebrew University Of Jerusalem-Free PDF

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YJSBI 5784 No of Pages 9 Model 5G,17 April 2010,ARTICLE IN PRESS. 2 F Mancia J Love Journal of Structural Biology xxx 2010 xxx xxx. nation of a membrane protein Detergents and their micelles are 125. notorious poor substitutes of lipids and their bilayer structures of 126. ten leading to destabilization denaturation and aggregation 127. Wiener 2004 Unfortunately detergents are required to extract 128. and purify the target protein The choice of detergent is a key 129. parameter of the entire process further complicated by the fact 130. that the shorter the aliphatic chain of the detergent the more 131. destabilizing the effect on the protein but the better becomes the 132. probability of crystallization and X ray diffraction to high resolu 133. tion Furthermore a detergent required for high yield extraction 134. may not be optimal in preserving functionality or oligomeric state 135. and may also have a detrimental impact on crystallization Wiener 136. 2004 Therefore different detergents may be required for the var 137. ious distinct phases of the necessary processes leading to structure 138. determination Extensive screening and optimization steps are 139. thus required These tedious procedures are time consuming and 140. expensive due the cost of reagents and the success rate is inevita 141. bly low 142, How can the probability of success for membrane protein struc 143. tures be maximized Following a conventional approach one 144. could envision optimizing expression extraction and puri cation 145. conditions in a tailor made approach to maximize yields and sta 146. bility of the given protein without any or with minimal interven 147. tion on the gene Structures of membrane proteins isolated from 148. Fig 1 Schematic representation of the NYCOMPS HT pipeline for the identi cation natural sources are inevitably con ned to being pursued by this 149. of prokaryotic membrane proteins suitable for structural studies Dapproach Alternatively the expression and puri cation protocols 150. can be xed and genetic variants of a protein of interest cloned 151. and screened to select the subset bearing the highest probability 152. 87 standard non af nity based puri cation methods such as ion ex of crystallization Here expression conditions are set to maximize 153. 88 change and hydrophobic interaction chromatography Membrane yields and chosen based on experience and consensus Puri cation 154. 89 proteins have the tendency to perform poorly in ectopic expression parameters are de ned to select expressing proteins that abide to 155. 90 systems Grisshammer and Tate 1995 This may be caused by a ser one or more characteristics thought to be indicative suggestive of 156. 91 ies of reasons including toxicity of the foreign protein to the host or necessary for successful crystallization These include for exam 157. 92 speci c requirements or differences in the translocation membrane ple a sharp elution pro le from size exclusion chromatography 158. 93 insertion machinery or in the lipid composition of the membranes SEC or stability in a short chain detergent Lemieux et al 2003 159. 94 Mancia and Hendrickson 2007 and possibly the limited amount Homology screening is a powerful approach and is the basis of 160. 95 of free surface available for packing large amounts of recombinant many structural genomics initiatives Homologues provide natural 161. 96 protein The situation is further aggravated for eukaryotic mem variation that may be less toxic better expressed more stable par 162. 97 brane proteins given the complexity of the milieu in which they ticularly in short chain detergents and may even be more likely to 163. 98 evolved and their frequent need for chaperones for speci c lipids crystallize Screening can also be performed on synthetic variants 164. 99 and for precise post translational modi cations such as glycosyla such as mutants with the goal of selecting those with enhanced 165. 100 tion sulfonation palmitoylation and other covalent modi cations properties like expression levels and thermal stability Serrano 166. 101 Mancia and Hendrickson 2007 Mancia et al 2004 In addition Vega et al 2008 Warne et al 2009 167. 102 post translational modi cations add possible heterogeneity to the A high throughput HT screening platform is essential for this 168. 103 sample which may hinder downstream crystallization success Typ approach to succeed For soluble proteins this has been achieved 169. 104 ically prokaryotic membrane proteins are expressed in Escherichia and suitable robust platforms extensively tested and thoroughly 170. 105 coli although systems based on Lactococcus lactis have been devel optimized leading to the determination of thousands of high res 171. 106 oped and successfully employed Kunji et al 2003 The success of olution structures Dessailly et al 2009 Joachimiak 2009 Fur 172. 107 E coli is due to the fact that it is robust economical well understood thermore technologies developed by structural genomics centers 173. 108 readily expandable and easily manipulated for labeling of selected have also been invaluable to the community at large and on aver 174. 109 amino acids such as methionine with selenium for phasing of age costs associated with every structure determination have 175. 110 diffraction data from crystals Hendrickson 1991 Eukaryotic coun dropped dramatically Terwilliger et al 2009 176. 111 terparts with notable exceptions Grisshammer and Tate 1995 In the use of HT methodologies membrane proteins are once 177. 112 Mancia and Hendrickson 2007 Grisshammer et al 1993 are again lagging behind their soluble counterparts However several 178. 113 dependent for their successful expression on matching or similarly reports on every stage of the process have facilitated the design 179. 114 complex systems such as yeast baculovirus infected insect cells of platforms and the adaptation of HT techniques for membrane 180. 115 and mammalian cells Mancia et al 2004 Midgett and Madden protein cloning expression screening and puri cation 181. 116 2007 In general eukaryotic membrane proteins have been recalci In an excellent review of collective methods used in the expres 182. 117 trant in expression at the scale needed for structural analysis sion and puri cation of over 10 000 soluble proteins Graslund 183. 118 Mancia and Hendrickson 2007 although high resolution struc et al 2008 a set of consensus protocols were presented and some 184. 119 tures from recombinant sources are nally emerging at a much of these procedures could be adaptable to the HT production of 185. 120 needed accelerating pace for examples see Gonzales et al 2009 membrane proteins This is particularly true for the initial cloning 186. 121 Hanson and Stevens 2009 Kawate et al 2009 Long et al 2005 steps where ligation independent cloning LIC Aslanidis and de 187. 122 Sobolevsky et al 2009 Tao et al 2009 Jong 1990 is very useful as it is rapid economical and ef cient 188. 123 Choice and optimization of a suitable expression system are by it requires no restriction digestion of the PCR ampli ed product 189. 124 no means the only requirements for successful structure determi and also can be designed to include no additional amino acids in 190. Please cite this article in press as Mancia F Love J High throughput expression and puri cation of membrane proteins J Struct Biol 2010. doi 10 1016 j jsb 2010 03 021,YJSBI 5784 No of Pages 9 Model 5G. 17 April 2010,ARTICLE IN PRESS, F Mancia J Love Journal of Structural Biology xxx 2010 xxx xxx 3. 191 the transcript LIC has been successfully used for large scale mem method for likelihood of crystallization of membrane proteins 257. 192 brane protein cloning efforts Clark et al xxxx Wang et al 2003 as it is for soluble proteins Klock et al 258. 193 For HT expression the consensus points to E coli being the pre 2008 The elution pro le of a membrane protein from a SEC 259. 194 ferred host for prokaryotic proteins although L lactis Kunji et al column equilibrated in a given detergent can provide a reliable 260. 195 2003 and cell free systems and have also been proposed Liguori estimate on their aggregation state and in general on their 261. 196 et al 2007 Schwarz et al 2008 Economical small scale incuba well being in that surfactant SEC can be readily adapted to HT 262. 197 tors have been developed that are useful for high density growth methods with micro volume HPLCs tted with autoloaders and 263. 198 of E coli in deep well blocks Optical densities measured at appropriately sized columns It can also be given added value by 264. 199 600 nm approaching 10 units are possible with just 600 mL of cul being coupled to static light scattering and refractive index detec 265. 200 ture Page and R 2004 Cultures grown under these conditions tors allowing quantitative evaluation of excess detergent micelle 266. 201 appear also to be scalable with growth in both specialized 96 tube size and aggregation state Veesler et al 2009 SEC analysis can 267. 202 airlift fermenters Lesley et al 2002 and ultra asks Brodsky and be streamlined further by eliminating other time consuming puri 268. 203 Cronin 2006 cation steps and using an in line uorescence detector to moni 269. 204 Determination of optimal expression extraction and puri ca tor the elution pro les of GFP fusions directly from miniscule 270. 205 tion parameters has been the focus of several studies on varying amounts of detergent solubilized cell extracts Kawate and Gou 271. 206 numbers of membrane protein targets ranging from several tens aux 2006 Fluorescence can also be used to estimate expression 272. 207 to few hundreds In two manuscripts Dobrovetsky et al outline a levels of GFP membrane protein fusions as shown by Hannon 273. 208 HT process for membrane protein expression utilizing a single et al on of 300 proteins from 18 bacterial and archeal extremo 274. 209 af nity tag and promoter system one extraction and puri cation philes Hammon et al 2009 The authors nd that after bench 275. 210 detergent followed by ion exchange chromatography or SEC as a marking levels of uorescence from the fusion tag to levels of 276. 211 nal puri cation step Dobrovetsky et al 2005 Dobrovetsky et al target protein expression they can easily detect membrane pro 277. 212 2007 Using these techniques they were able to screen 280 teins produced in amounts suitable for structural studies The only 278. 213 E coli and Thermotoga maritima integral membrane proteins The requirements are that the terminus of the protein that the GFP is 279. 214 authors conclude that in a manner analogous to the techniques in attached to remains in the reducing environment of the cytoplasm 280. 215 volved in HT platforms for soluble proteins similar approaches for so that the GFP uorescence develops properly Daley et al 2005 281. 216 membrane proteins will succeed but with higher attrition rates This may render a percentage of membrane proteins not amenable 282. Lewinson et al investigate many of the necessary parameters nec. essary of a structural genomics type approach to the production of. Dto this technique In addition it may still be necessary to perform. standard SDS PAGE analysis to verify that the uorescence signal. 219 membrane proteins Lewinson et al 2008 focusing on the pro recorded is from the intact full length fusion rather than from 285. 220 karyotic P type transporter family Yatime et al 2009 The truncation products 286. 221 authors set out to express and purify multiple homologues in mul The studies reviewed above and our own experience allowed us 287. 222 tiple strains temperatures af nity tags promoters and extraction to construct an expression and screening platform for prokaryotic 288. 223 and puri cation detergents They nd that many factors appear to membrane proteins presented here 289. 224 impact the nal outcome but conclude that i the closer phylum. 225 of the target gene to the expression host the better the possible. 226 outcome ii the promoter may affect the amount of protein pro 2 Materials and methods 290. 227 duced and the membrane incorporation levels iii the position. 228 and nature of the af nity tag may have a profound impact on 2 1 Target selection protocol 291. 229 expression levels iv that only a small subset of extraction deter. 230 gents results in a high percentage of success in solubilizing a In depth discussion and experimental details for the target 292. 231 majority of the membrane proteins with n dodecyl b D maltopyr selection process can be found in Punta et al 2009 The initial NY 293. 232 anoside DDM being the most favored In a similar study by Esha COMPS target set comprised more than 300 000 annotated se 294. 233 ghi et al the expression pro le of 49 E coli membrane proteins was quences from the RefSeq collection Pruitt et al 2007 belonging 295. 234 analyzed in this host varying tag position expression strain and to 96 fully sequenced prokaryotic genomes Since most targets 296. 235 extraction detergent Eshaghi et al 2005 Gateway Hartley have no experimental annotation linking them to the membrane 297. 236 et al 2000 was utilized for the cloning which may not be ideal the prediction program TMHMM2 Krogh et al 2001 was used 298. 237 for structural studies due to the introduction of additional amino to predict transmembrane helices TMHs Although prediction 299. 238 acids at the protein termini These parameters were assessed for methods are estimated to be very accurate they will inevitably 300. 239 success by dot blot and gel ltration analysis of some of the targets make helix prediction mistakes Therefore we retained only pro 301. membrane protein production and preliminary characterization 61 that would achieve a comparable level of productivity to that 62 observed for soluble proteins in the initial phases of the Protein 63 StructureInitiative PSI Toful llthisgoal wedesignedandimple 64 mented a centralized Protein Production Facility at the New York 65

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