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

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

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

¨
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 ().
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

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

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

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.
). 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].

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 () entry is indicated in parentheses, where applicable.
b
Expression and

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

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

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 ⁄ 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

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

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

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

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. 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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