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’ PrPC in healthy organisms is i
Trang 1structural 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’ (PrPC) 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 PrPCto 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 glycosylaforma-tion 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 PrPCs 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; PrPC, cellular form of prion protein
Trang 2As 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
PrPCs 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 unsucsuc-cessful 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 nonmammamamma-lian species, which have been
pre-pared for studies on the structure and function of
PrPC, 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 13C,15N-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 concon-structs 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 PrPC 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 PrPC, 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 PrPC 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),
Trang 3Table 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 PrPC pRSET A is a vector obtained from Invitrogen
Species
Accession no
Construct cloned
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]
refolded soluble protein fPrP(121–231)b,c NMR structure solved (1XYJ) [11]
bPrP(90–230)b High-yield expression, high yield of
refolded soluble protein
refolded soluble protein chPrP(121–225)b,c NMR structure solved (1U3M) [13]
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
Fugu (Takifugu rubripes) AAN38988 Fugu-PrP1(298–423)d High-yield expression, reconstitution
yielded a nonglobular polypeptide [24]
refolded soluble protein ecPrP(121–231)b,c NMR structure determination in progress
hPrP(81–230)b High-yield expression, high yield of
refolded soluble protein
hPrP(121–230)b NMR structure solved (1QM3, 1HJN) [10,20]
hPrP(130–230)b High-yield expression, high yield of
refolded soluble protein
hPrP(121–226)b High-yield expression, high yield of
refolded soluble protein
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 datae
Trang 4which 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 PrPC, 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 PrPC
[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
hPrP[E219Q](121–230)b High-yield expression,
high yield of refolded soluble protein hPrP[R220K](121–230)b NMR structure solved
(1E1U) [19]
refolded soluble protein scPrP(121–231)b,c NMR structure solved (1XYQ) [11]
ovPrP[Q168R](121–231)b,c NMR structure solved (1Y2S) [11] Tammar wallaby
(Macropus eugenii)
twPrP(121–235)b,c High-yield expression, reconstitution
yielded a nonglobular polypeptide
refolded soluble protein tPrP(121–225)b,c NMR structure solved (1U5L) [13]
aSurvey of the protein biochemistry; the PDB (http://www.rcsb.org) entry is indicated in parentheses, where applicable.bExpression and purification as described in Zahn et al [10,18].cPurification as described in Lysek & Wu¨thrich [23].dNumeration according to Fugu PrP1 [39].eEither initial expression attempts were not successful, or successful expression was not followed up, or no expression trials were started.fHuman doppel protein
Trang 5Table 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)
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 dataf
mPrP[S170N⁄ N174T](23–231)c,d High-yield expression, high yield of refolded soluble protein
mPrP[Y225A⁄ Y226A](23–231)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 dataf
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 dataf
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
mPrP[P105L](91–231)c,d High-yield expression, high yield of refolded soluble protein
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
GB1-mPrP(104–231)c,d,g High-yield expression, high yield of refolded soluble protein
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 dataf
GB1-mPrP[A113V⁄ A115V ⁄ A118V](104–231)g No expression dataf
mPrP[A113V⁄ A115V ⁄ A118V](109–231) No expression dataf GB1-mPrP[A113V⁄ A115V ⁄ A118V](109–231)c,d,g High-yield expression, high yield of refolded soluble protein
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 ⁄ D227E](121–231) No expression dataf
mPrP[V166G](121–231)c,d High-yield expression, low yield of refolded soluble protein
Trang 6Table 2 (Continued)
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[Q168E⁄ N173K](121–231)c,d High-yield expression, high yield of refolded soluble protein
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 dataf
mPrP[Y169F⁄ F175A](121–231)c,d High-yield expression, high yield of refolded soluble protein
mPrP[Y169G⁄ S170N ⁄ N174T](121–231)c,d,e
High-yield expression, high yield of refolded soluble protein
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[N173K](121–231)c,d High-yield expression, high yield of refolded soluble protein
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
GB1-mPrP[K110I⁄ H111I](90–130)g
No expression dataf
Trang 7optimized 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 chromatogchromatog-raphy, and gel filtration,
with yields of 5–10 mg of pure protein per liter of rich
med-ium and 2–3 mg from minimal medmed-ium 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 CuSO4 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 Ni2+–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
Ni2+–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)
GB1-mPrP[A113V⁄ A115V ⁄ A118V](90–130)g No expression dataf
GB1-mPrP(90–140)c,d,g High-yield expression, reconstitution yielded a nonglobular polypeptide
GB1-mPrP[K110I⁄ H111I](90–140)g No expression dataf
mPrP[A113V⁄ A115V ⁄ A118V](90–140) No expression dataf
GB1-mPrP[A113V⁄ A115V ⁄ A118V](90–140)g
No expression dataf 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.bExpression and purification as described in Hornemann et al [15].cExpression and purification as described in Zahn et al [10,18].dPurification as described
in Lysek & Wu¨thrich [23].eSee Experimental procedures for the different purification protocol used for these PrP variants.fEither initial expression attempts were not successful, or successful expression was not followed up, or no expression trials were started.gGB1 stands for the 56 residue B1 immunoglobulin-binding domain of streptococcal protein G [32,33].hExpression and purification as described in Horne-mann & Glockshuber [36].iMouse doppel protein (NCBI accession number AAF02544).jMouse Shadoo protein (NCBI accession number
NP 898970)
Trang 8National 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 Schorn and
R Zahn contributed expression plasmids to the
collec-tions in Tables 1 and 2 (for details, see the references
given in the tables).
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