Prion protein library of recombinant constructs for structural biology Simone Hornemann* , à, Barbara Christenà, Christine von Schroetter, Daniel R. Pe ´ rez and Kurt Wu ¨ thrich Institute of Molecular Biology and Biophysics, ETH Zurich, Switzerland Expression of the prion protein (PrP) in its ‘cellular form’ (PrP C ) in healthy organisms is intimately related to susceptibility to transmissible spongiform encephal- opathies, such as scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt–Jakob disease in humans [1]. Transmissible spongiform encephalopathies are related to the conver- sion of PrP C to a protease-resistant b-sheet-rich ‘scrapie form’ [2]. The gene encoding PrP has been sequenced [3,4], and post-translational modifications, such as cleavage of N-terminal and C-terminal signal sequences during import into the endoplasmic reticulum, forma- tion of a disulfide bond, N-linked glycosylation at two sites, and addition of a C-terminal glycosylphosphat- idylinositol anchor, have been described [5,6]. Structure determinations by NMR spectroscopy have shown that PrP C s from mammals, birds, reptiles and amphibians all possess the same molecular architecture, consisting of a flexibly extended 100 residue N-terminal tail and a globular C-terminal domain of similar size [7–14]. Keywords NMR structure determination; prion protein plasmid library; prion protein structural biology; recombinant prion proteins; transmissible spongiform encephalopathies Correspondence K. Wu ¨ thrich, Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland Fax: +41 44 633 1151 Tel: +41 44 633 2473 E-mail: wuthrich@mol.biol.ethz.ch Website: http://www.mol.biol.ethz.ch/ groups/wuthrich_group *Present address Institute of Neuropathology, Universita ¨ ts- Spital Zurich, Switzerland àThese authors contributed equally to this work (Received 23 December 2008, revised 12 February 2009, accepted 13 February 2009) doi:10.1111/j.1742-4658.2009.06968.x A survey of plasmids for 51 prion protein constructs from bank vole, cat, cattle, chicken, dog, elk, ferret, frog, fugu, horse, human, pig, sheep, turtle, and wallaby, and for 113 mouse prion protein constructs and variants thereof, is presented. This includes information on the biochemistry of the recombinant proteins, in particular on successful and unsuccessful expres- sion attempts. The plasmid library was generated during the past 12 years in the context of NMR structure determination and biophysical character- ization of prion proteins in our laboratory. The plasmids are now available for general use, and are distributed free of charge to not-for-profit institutions. Abbreviations hPrP, human prion protein; mPrP, mouse prion protein; PrP, prion protein; PrP C , cellular form of prion protein. FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS 2359 As part of a major project on PrP structural biology pursued over the past 12 years, our laboratory has generated recombinant constructs of the mature forms of PrPs from a variety of mammalian and nonmamma- lian species, and of partial sequences thereof. In addi- tion, designed variants of mouse PrP (mPrP) and human PrP (hPrP) were prepared; these include mimics of most of the pathological mutations identified in hPrPs and a selection of variant PrPs observed in other species. Some of these constructs have previously been described in connection with structural studies of PrP C s by NMR spectroscopy [7,9–24], and in reports on physical–chemical studies, such as transformation into insoluble fibrils [25]. A large number of additional PrP constructs have been cloned, and in part also expressed and purified for extensions of and as internal controls in our studies, without being explicitly described in earlier publications. In view of the contin- ued widespread interest in exploring the role of the PrP in health and disease (see above), and considering that the PrP constructs generated in our laboratory could be of use to others for functional or further structural studies, this article presents a survey of all the PrP plasmids available from us upon request, and provides a concise account of our experience with the biochemistry of recombinant PrPs, in particular of suc- cessful as well as unsuccessful expression attempts. Results and Discussion This section presents listings of plasmids that encode the mature forms, devoid of the signal sequences, of natural and modified PrPs from a variety of mamma- lian and nonmammalian species, which have been pre- pared for studies on the structure and function of PrP C , and which are now available upon request for use elsewhere. The data are collected in Tables 1 and 2, and at the end of this section we provide informa- tion on where the plasmids can be obtained. All plas- mids were designed for protein expression in bacterial cultures and not for expression in mammalian cells. Table 1 lists plasmids encoding tetrapod and fish PrP sequences. This includes columns containing the binomial name of the species, the accession number of the sequence in the NCBI protein database, the con- struct length, and information on the biochemical investigations performed. The summary statements in the last column have the following meaning: ‘NMR structure solved’ indicates that stable solutions con- taining about 1 mm concentrations of 13 C, 15 N-labeled protein were obtained from cultures in minimal medium; the Protein Data Bank (PDB) entry code and literature references are given. ‘NMR structure determination in progress’ has the same meaning, except that PDB deposition and publication are still in progress. For all other constructs no NMR structure determination has been performed, either because this would not have been of interest in the context of the ongoing projects, or because of the lack of sufficient amounts of purified protein. The indications of the yields of expression and reconstitution for these con- structs are self-explanatory, whereby the constructs with high yields of natively refolded soluble protein can be considered as promising candidates for future NMR structure determinations, or for other studies that require milligram amounts of pure protein with long-term stability of the PrP C form. The term ‘no expression data’ is used if either the initial expression trials were unsuccessful, or initial successful expression was not followed up, or no expression trials have been performed. For most species, two PrP plasmids are listed: a first one encoding the polypeptide corresponding to the ‘full-length’ mature PrP C , usually comprising residues 23–231, and a second one encoding a C-terminal frag- ment spanning residues 121–231 (see Scha ¨ tzl et al. [26] for the numeration used in this article). This C-terminal fragment forms a globular domain both as part of the full-length sequence and in the isolated form, and there- fore constructs of the isolated C-terminal globular domain have been used for NMR structure determina- tions with many of the species [7,9–14,19,20,23,24]. Actually, structure determinations of the full-length protein have been performed only for the PrPs from mouse (Table 2), cattle, and humans. For hPrP, Table 1 also lists a number of designed variants of hPrP(121– 230), most of which have been inspired by natural varia- tions in mammalian PrP amino acid sequences. Human PrP fragments of variable lengths have been used to study the minimal length of the amino acid sequence that is needed for stability of the globular domain fold (R. Zahn, C. von Schroetter & K. Wu ¨ thrich, unpub- lished results). Finally, the human doppel protein has also been included in Table 1. Mouse PrP was used as a reference in most of our projects. For example, whenever the PrP C structure from a different species displayed significant local dif- ferences when compared to mPrP, selected single amino acid replacements, or combinations thereof, were introduced into mPrP to search for the sequence features that cause the local variations in the three- dimensional structure. A large number of constructs were thus derived from the mPrP sequence, and these are given in Table 2, where they are listed in order of decreasing chain length. Overall, Table 2 is dominated by a large number of variants of mPrP(121–231), Plasmid library of prion proteins S. Hornemann et al. 2360 FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS Table 1. List of plasmids encoding the sequence of the mature cellular form of the prion protein from a variety of species and truncated variants thereof, and of human doppel. The protein accession number refers to the NCBI protein database (http://www.ncbi.nlm.nih.gov). For the American elk, bank vole, chicken, dog, ferret, pig, sheep, tammar wallaby, and turtle, the reference is for the C-terminal sequence fragment that forms a globular domain in PrP C . pRSET A is a vector obtained from Invitrogen. Species Accession no. Construct cloned into pRSET A Comments (PDB) a American elk (Cervus elaphus nelsoni) AAB94788 ePrP(23–230) b,c High-yield expression, high yield of refolded soluble protein ePrP(121–230) b,c NMR structure solved (1XYW) [12] Bank vole (Clethrionomys glareolus) AAL57231 bvPrP(121–231) b,c NMR structure solved (2K56) [14] Cat (Felis catus) AAS94127 fPrP(23–231) b,c High-yield expression, high yield of refolded soluble protein fPrP(121–231) b,c NMR structure solved (1XYJ) [11] Cattle (Bos taurus) ABU97893 bPrP(23–230) b NMR structure solved (1DX1) [9] bPrP(90–230) b High-yield expression, high yield of refolded soluble protein bPrP(121–230) b NMR structure solved (1DWZ) [9] Chicken (Gallus gallus) NP_990796 chPrP(23–225) b,c High-yield expression, high yield of refolded soluble protein chPrP(121–225) b,c NMR structure solved (1U3M) [13] Dog (Canis familiaris) AAD12061 cPrP(23–231) b,c High-yield expression, high yield of refolded soluble protein cPrP(121–231) b,c NMR structure solved (1XYK) [11] Ferret (Mustela putorius furo) AAA69022 Ferret PrP (121–231) b,c High-yield expression, reconstitution yielded a nonglobular polypeptide Frog (Xenopus laevis) AAV54126 xlPrP(90–222) b,c NMR structure solved (1XU0) [13] Fugu (Takifugu rubripes) AAN38988 Fugu-PrP1(298–423) d High-yield expression, reconstitution yielded a nonglobular polypeptide [24] Horse (Equus caballus) ABL86003 ecPrP(23–231) b,c High-yield expression, high yield of refolded soluble protein ecPrP(121–231) b,c NMR structure determination in progress Human (Homo sapiens) P04156 hPrP(23–230) b NMR structure solved (1QLZ) [10,38] hPrP(81–230) b High-yield expression, high yield of refolded soluble protein hPrP(90–230) b NMR structure solved (1QM1) [10] hPrP(96–230) No expression data e hPrP(100–230) No expression data e hPrP(105–230) No expression data e hPrP(110–230) No expression data e hPrP(121–230) b NMR structure solved (1QM3, 1HJN) [10,20] hPrP(126–230) No expression data e hPrP(130–230) b High-yield expression, high yield of refolded soluble protein hPrP(135–230) No expression data e hPrP(23–222) No expression data e hPrP(23–226) No expression data e hPrP(121–226) b High-yield expression, high yield of refolded soluble protein hPrP(121–222) No expression data e hPrP(126–219) No expression data e hPrP(126–222) No expression data e hPrP(126–226) No expression data e hPrP[M166C ⁄ E221C](121–230) b NMR structure solved (1H0L) [22] hPrP[M166V](121–230) b NMR structure solved (1E1G) [19] hPrP[S170N](121–230) b NMR structure solved (1E1P) [19] hPrP[I215V](121–230) b High-yield expression, high yield of refolded soluble protein hPrP[Q217R](121–230) No expression data e S. Hornemann et al. Plasmid library of prion proteins FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS 2361 which contain single or multiple amino acid replace- ments relative to the wild-type sequence. Many of these sequence variations are located in a surface epitope formed by the polypeptide segment 165–175, which forms a loop that connects a b-strand with an a-helix in PrP C , and the polypeptide segment 220–228, which forms part of a C-terminal a-helix [12,14]. The extremely high variability in both the sequence and local conformation of this epitope [27,28] has attracted special interest with regard to the physiologi- cal role [29–31] and the structural biology of PrP C [12,14]. Additional plasmids listed in Table 2 encode full- length mPrP and constructs containing residues 90–231 or shorter fragments of the mPrP sequence. A selection of the amino acid replacements studied in mPrP(121– 231) was also introduced into constructs of different lengths, e.g. to obtain internal controls for their effects on the three-dimensional structure. Constructs with amino acid exchanges outside of the globular domain were used to study the effects of sequence variations on interactions with membrane mimics, such as deter- gent micelles (S. Hornemann, C. von Schroetter, F. F. Damberger & K. Wu ¨ thrich, unpublished results), or on conformational equilibria. For some of these projects, the N-terminal fusion tag GB1 [32,33] was added to the constructs in order to enhance the expres- sion yield and the solubility of selected mPrP constructs. Finally, Table 2 also includes the mouse doppel protein, for which the NMR solution structure has been determined by Mo et al. [34], and the mouse Shadoo protein, which has recently been biochemically characterized [35]. The plasmids listed in Tables 1 and 2 are available free of charge for use in academic and other not-for- profit institutions by contacting S. Hornemann at Uni- versita ¨ tsSpital Zurich, Institute of Neuropathology, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland (simone. hornemann@usz.ch). We will not be in a position to entertain requests either for crude cell extracts or for purified proteins. Experimental procedures The procedures used in our laboratory for the cloning, expression and purification of recombinant PrPs have been developed mainly with full-length and truncated constructs of mPrP and hPrP. Here, we present short descriptions of these procedures as they were applied to prepare the proteins of Tables 1 and 2 [10,15,18,23,36,37]. mPrP(121–231) from soluble expression in Escherichia coli periplasmic extracts using the vector pPrP-CRR The gene that encodes for mPrP(121–231) was fused to the bacterial OmpA signal sequence for secretory periplasmic expression, yielding the expression vector pPrP-CRR Table 1. (Continued) Species Accession no. Construct cloned into pRSET A Comments (PDB) a hPrP[E219Q](121–230) b High-yield expression, high yield of refolded soluble protein hPrP[R220K](121–230) b NMR structure solved (1E1U) [19] Q9UKY0 f hDpl(24–152) b,f NMR structure solved (1LG4) [21] Pig (Sus scrofa) AAA92862 scPrP(23–231) b,c High-yield expression, high yield of refolded soluble protein scPrP(121–231) b,c NMR structure solved (1XYQ) [11] Sheep (Ovis aries) AAC78726 ovPrP[Q168H](121–231) b,c NMR structure solved (1XYU) [11] ovPrP[Q168R](121–231) b,c NMR structure solved (1Y2S) [11] Tammar wallaby (Macropus eugenii) AAT68001 twPrP(121–230) b,c NMR structure determination in progress twPrP(121–235) b,c High-yield expression, reconstitution yielded a nonglobular polypeptide Turtle (Trachemys scripta) CAB81568 tPrP(23–225) b,c High-yield expression, high yield of refolded soluble protein tPrP(121–225) b,c NMR structure solved (1U5L) [13] a Survey of the protein biochemistry; the PDB (http://www.rcsb.org) entry is indicated in parentheses, where applicable. b Expression and purification as described in Zahn et al. [10,18]. c Purification as described in Lysek & Wu ¨ thrich [23]. d Numeration according to Fugu PrP1 [39]. e Either initial expression attempts were not successful, or successful expression was not followed up, or no expression trials were started. f Human doppel protein. Plasmid library of prion proteins S. Hornemann et al. 2362 FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS Table 2. List of plasmids encoding the sequence of the mature cellular form of mPRP (accession number AAA39997) and truncated forms and designed variants thereof, and of mouse doppel and Shadoo protein constructs. Protein accession numbers refer to the NCBI database (http://www.ncbi.nlm.nih.gov). pRSET A is a vector obtained from Invitrogen. mPrP(23–231) is also available in the vector pRBI-PDI-T7, and mPrP(121–231) in the vector pPrP-CRR (see Experimental procedures). Segment Construct cloned into pRSET A Comments (PDB) a 23–231 mPrP(23–231) b,c,d NMR structure solved [16] mPrP[K110I ⁄ H111I](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[A113V ⁄ A115V ⁄ A118V](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ N173K](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169G ⁄ S170N ⁄ N174T](23–231) c,d,e High-yield expression, high yield of refolded soluble protein mPrP[S170N](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[S170N ⁄ N171G ⁄ N174T](23–231) No expression data f mPrP[S170N ⁄ N174T](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y225A](23–231) No expression data f mPrP[Y225A ⁄ Y226A](23–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y226A](23–231) No expression data f 90–231 mPrP(90–231) c,d High-yield expression, high yield of refolded soluble protein GB1-mPrP(90–231) c,d,g High-yield expression, high yield of refolded soluble protein mPrP[K110I ⁄ H111I](90–231) c,d High-yield expression, high yield of refolded soluble protein GB1-mPrP[K110I ⁄ H111I](90–231) g No expression data f mPrP[H111A ⁄ A117V](90–231) No expression data f mPrP[A113V ⁄ A115V ⁄ A118V](90–231) c,d High-yield expression, high yield of refolded soluble protein GB1-mPrP[A113V ⁄ A115V ⁄ A118V](90–231) g No expression data f mPrP[A117V](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[A117V ⁄ M129V](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ N173K](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169A](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169G ⁄ S170N ⁄ N174T](90–231) c,d,e High-yield expression, high yield of refolded soluble protein mPrP[S170N](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[S170N ⁄ N174T](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[N174T](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[F175A](90–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y225A ⁄ Y226A](90–231) c,d High-yield expression, high yield of refolded soluble protein 91–231 mPrP[P102L](91–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[P105L](91–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[P105L ⁄ H111A](91–231) No expression data f mPrP[P105L ⁄ M129V](91–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[M129V](91–231) c,d High-yield expression, high yield of refolded soluble protein 104–231 mPrP(104–231) c,d High-yield expression, high yield of refolded soluble protein GB1-mPrP(104–231) c,d,g High-yield expression, high yield of refolded soluble protein mPrP[K110I ⁄ H111I](104–231) No expression data f GB1-mPrP[K110I ⁄ H111I](104–231) c,d,g High-yield expression, high yield of refolded soluble protein mPrP[A113V ⁄ A115V ⁄ A118V](104–231) No expression data f GB1-mPrP[A113V ⁄ A115V ⁄ A118V](104–231) g No expression data f 109–231 mPrP(109–231) No expression data f GB1-mPrP(109–231) g No expression data f mPrP[A113V ⁄ A115V ⁄ A118V](109–231) No expression data f GB1-mPrP[A113V ⁄ A115V ⁄ A118V](109–231) c,d,g High-yield expression, high yield of refolded soluble protein 121–231 mPrP(121–231) c,d,h NMR structure solved (1AG2, 1XYX) [7,12] GB1-mPrP(121–231) c,d,g High-yield expression, high yield of refolded soluble protein mPrP[R148H](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y155N ⁄ S170N](121–231) No expression data f mPrP[Y155N ⁄ S170N ⁄ D227E](121–231) No expression data f mPrP[V166A](121–231) c,d NMR structure determination in progress mPrP[V166G](121–231) c,d High-yield expression, low yield of refolded soluble protein mPrP[D167S](121–231) c,d NMR structure determination in progress S. Hornemann et al. Plasmid library of prion proteins FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS 2363 Table 2. (Continued) Segment Construct cloned into pRSET A Comments (PDB) a mPrP[D167S ⁄ Q168E ⁄ N173K](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169A ⁄ S170N ⁄ N173K ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169A ⁄ S170N ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169A ⁄ N173K](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169F](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169F ⁄ S170N ⁄ N173K ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169F ⁄ S170N ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ Y169G ⁄ N173K](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ S170N ⁄ N173K ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ S170N ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D167S ⁄ N173K](121–231) c,d NMR structure determination in progress mPrP[D167S ⁄ N173K ⁄ E221A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[D168E ⁄ Y169A](121–231) No expression data f mPrP[Q168E ⁄ N173K](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169A](121–231) c,d NMR structure determination in progress mPrP[Y169A ⁄ S170N ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169A ⁄ F175A](121–231) c,d High-yield expression, reconstitution yielded a nonglobular polypeptide mPrP[Y169A ⁄ Y225A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169A ⁄ Y225A ⁄ Y226A](121–231) c,d NMR structure determination in progress mPrP[Y169F](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169F ⁄ S170N ⁄ N174T](121–231) No expression data f mPrP[Y169F ⁄ F175A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y169G](121–231) c,d NMR structure determination in progress mPrP[Y169G ⁄ S170N ⁄ N174T](121–231) c,d,e High-yield expression, high yield of refolded soluble protein mPrP[S170N](121–231) c,d NMR structure solved (2K5O) [14] mPrP[S170N ⁄ N171A ⁄ N174T](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[S170N ⁄ N171G ⁄ N174T](121–231) c,d NMR structure determination in progress mPrP[S170N ⁄ N174T](121–231) c,d NMR structure solved (1Y16) [12] mPrP[N173K](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP(N174T)(121–231) c,d NMR structure solved (1Y15) [12] mPrP[F175A](121–231) c,d NMR structure determination in progress mPrP[F175A ⁄ Y218A](121–231) c,d High-yield expression, reconstitution yielded a nonglobular polypeptide mPrP[F175A ⁄ Y218F](121–231) c,d High-yield expression, low yield of refolded soluble protein mPrP[F175A ⁄ Y225A ⁄ Y226A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[F175L](121–231) c,d High-yield expression, reconstitution yielded a nonglobular polypeptide mPrP[D178A](121–231) c,d High-yield expression, low yield of refolded soluble protein mPrP[D178N](121–231) c,d High-yield expression, low yield of refolded soluble protein mPrP[Y218A](121–231) c,d High-yield expression, reconstitution yielded a nonglobular polypeptide mPrP[Y225A](121–231) c,d High-yield expression, high yield of refolded soluble protein mPrP[Y225A ⁄ Y226A](121–231) c,d NMR structure determination in progress mPrP[Y226A](121–231) c,d High-yield expression, high yield of refolded soluble protein 122–228 mPrP(122–228) No expression data f 122–230 mPrP(122–230) No expression data f 127–228 mPrP(127–228) No expression data f 127–230 mPrP(127–230) No expression data f 23–144 mPrP(23–144) No expression data f 90–130 mPrP(90–130) No expression data f GB1-mPrP(90–130) g No expression data f mPrP[K110I ⁄ H111I](90–130) No expression data f GB1-mPrP[K110I ⁄ H111I](90–130) g No expression data f mPrP[A113V ⁄ A115V ⁄ A118V](90–130) No expression data f Plasmid library of prion proteins S. Hornemann et al. 2364 FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS optimized for the most frequent Arg codons found in strongly expressed E. coli genes [36,37]. After expression in the periplasm of E. coli BL21 cells, native mPrP(121–231) was purified to homogeneity by anion exchange chromatog- raphy, hydrophobic chromatography, and gel filtration, with yields of 5–10 mg of pure protein per liter of rich med- ium and 2–3 mg from minimal medium. After dialysis against distilled water, the protein was stored at )20 °C. mPrP(23–231) from inclusion bodies expressed in E. coli cytoplasm using the vector pRBI-PDI-T7 The gene encoding mPrP(23–231) was cloned into the vector pRBI-PDI-T7 and expressed as insoluble inclusion bodies in the cytoplasm of E. coli BL21 cells under the control of the T7 promoter ⁄ operator sequence [15]. After washing and sol- ubilization of the inclusion bodies in 8 m urea, the protein was purified by cation exchange chromatography in the presence of urea, and oxidized at low concentrations by air oxygen in the presence of 1 lm CuSO 4 . The oxidized protein was purified by cation exchange chromatography under native conditions in the presence of protease inhibitors, and then dialyzed against distilled water and stored at )20 °C. The yields were about 5 mg of pure protein per liter of rich medium and 2.5 mg from minimal medium. PrPs from inclusion bodies expressed in E. coli cytoplasm using the vector pRSET A with an N-terminal histidine tag The proteins were expressed as inclusion bodies in the cyto- plasm of E. coli BL21 cells under the control of the T7 pro- motor, and purified, oxidized and refolded by affinity chromatography using Ni 2+ –nitrilotriacetic acid agarose resin. The N-terminal histidine tail was then cleaved using thrombin, and the thrombin was removed by ion exchange chromatography. The yields were 10–20 mg of protein per liter of rich bacterial culture and 5–10 mg from minimal medium. In a modification of this approach [10,18], the removal of thrombin by ion exchange chromatography was replaced by addition of p-aminobenzamidine Celite, fol- lowed by centrifugation at 3500 g to remove the Celite [23]. This is the current ‘standard procedure’, which was used with most of the proteins listed in Tables 1 and 2. For some proteins, increased yields were achieved with constructs con- taining the solubility enhancement tag GB1 [32,33], which were generated by cloning the GB1 domain via the NdeI and BamHI restriction sites prior to inserting the PrP frag- ments via the restriction sites BamHI and EcoRI. Purification of mPrP variants containing the substitutions Y169G, S170N, and N174T The three proteins mPrP(Y169G ⁄ S170N ⁄ N174T)(23–231), mPrP(Y169G ⁄ S170N ⁄ N174T)(90–231) and mPrP(Y169G ⁄ S170N ⁄ N174T)(121–231) could not be eluted from the Ni 2+ –nitrilotriacetic acid column when using the standard purification method [23]. They were therefore eluted with 8 m urea containing 500 mm imidazole and 100 mm sodium phosphate at pH 8.0. The proteins were then dialyzed against 10 mm sodium acetate at pH 4.5. After addition of 10 mm Tris ⁄ HCl, the pH was adjusted to 8.3 and the His-tag was removed as described in [23]. Acknowledgements This project was supported by the Swiss National Sci- ence Foundation and ETH Zurich through the Table 2. (Continued) Segment Construct cloned into pRSET A Comments (PDB) a GB1-mPrP[A113V ⁄ A115V ⁄ A118V](90–130) g No expression data f 90–140 mPrP(90–140) No expression data f GB1-mPrP(90–140) c,d,g High-yield expression, reconstitution yielded a nonglobular polypeptide mPrP[K110I ⁄ H111I](90–140) No expression data f GB1-mPrP[K110I ⁄ H111I](90–140) g No expression data f mPrP[A113V ⁄ A115V ⁄ A118V](90–140) No expression data f GB1-mPrP[A113V ⁄ A115V ⁄ A118V](90–140) g No expression data f mDpl(24–155) b,i High-yield expression, low yield of refolded soluble protein mSho(25–123) c,d,j High-yield expression, reconstitution yielded a nonglobular polypeptide mSho(68–123) c,d,j High-yield expression, reconstitution yielded a nonglobular polypeptide a Survey of the protein biochemistry; the PDB (http://www.rcsb.org) entry is indicated in parentheses, where applicable. b Expression and purification as described in Hornemann et al. [15]. c Expression and purification as described in Zahn et al. [10,18]. d Purification as described in Lysek & Wu ¨ thrich [23]. e See Experimental procedures for the different purification protocol used for these PrP variants. f Either initial expression attempts were not successful, or successful expression was not followed up, or no expression trials were started. g GB1 stands for the 56 residue B1 immunoglobulin-binding domain of streptococcal protein G [32,33]. h Expression and purification as described in Horne- mann & Glockshuber [36]. i Mouse doppel protein (NCBI accession number AAF02544). j Mouse Shadoo protein (NCBI accession number NP 898970). S. Hornemann et al. Plasmid library of prion proteins FEBS Journal 276 (2009) 2359–2367 ª 2009 The Authors Journal compilation ª 2009 FEBS 2365 National Centre of Competence in Research ‘Struc- tural Biology’, and by a grant from the European Union (UPMAN). S. Bonjour, L. Calzolai, V. Esteve- Moya, A. D. Gossert, F. Lo ´ pez Garcı ´ a, T. Lu ¨ hrs, D. A. Lysek, L. G. Nivon, Y. Scha ¨ rli, C. 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