Over-expression and purification of recombinant murine wild type and mutant prion proteins in E. coli
B. SURIYAMONGKOL 1,2, D. DUGGAN1,2, V. SEMENCHENKO3 and D.S. WISHART1,2,*
Departments of 1 Biological Sciences and 2 Computing Science, University of Alberta, Edmonton, Alberta T6G 2E9
3 NanoLife Sciences Group, National Institute for Nanotechnology, Edmonton, Alberta T6G 2M9
* Corresponding author Tel: +1-780-492-0383 Email address: david.wishart@ualberta.ca
ABSTRACT
Prion diseases are a family of fatal neurodegenerative disorders, which affect both humans and animals. In humans, this includes Gerstmann-Straussler-Scheinker (GSS), Fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Familial
transmission of these diseases is associated with mutations in the PrP gene. To investigate the molecular basis of these mutations, recombinant Syrian Hamster prion (residues 90-231 - the protease resistant fragment) containing seven different point
mutations (P102L, A117V, M129V, D178N, F198S, E200K and M129V/F198S) were over-expressed in E. coli BL21(DE3). The polypeptides were expressed as N-terminal Histidine-tagged fusion proteins and purified using a nickel-NTA agarose resin.
Methods for protein refolding, which include on-column refolding and the use of membrane dialysis, were developed according to properties of each of the mutant proteins. Production yields of the target proteins vary from 10 to 45 milligrams per one liter
of E. coli culture depending on types of the mutants. Currently, the isotopically labeled samples are being prepared and subsequently characterized by MS and NMR analysis for structural studies.
INTRODUCTION
Prion
diseases
or
Transmissible
Spongiform
Encephalopathies (TSEs) in humans and animals were
shown to be caused by structural conversion of cellular
prion protein to the infectious scrapie isoform (1).
Investigation into these structural changes of both wild
type and mutant prion molecules, therefore, rely on highvolume preparations of the prion proteins. In this study,
we develop methods to optimize the production and
purification of recombinant Syrian Hamster mutant prion
proteins in E. coli.
GENE CONSTRUCT PREPARATION
EXPRESSION LEVELS OF MUTANT PROTEINS
kDa
1
2
3
4
5
6
7
8
Figure 2. Coomassie blue stained gel image of cell lysates of E. coli BL21(DE3)
carrying wild type and mutant SHPrPc(90-231) constructs. Pilot expression was
performed using a small-volume culture (1 ml). Over-expression of recombinant prion
protein was induced by IPTG to a final concentration of 1 mM of the culture. Lysates in
lanes 1 to 8 showed intense protein bands (18 kDa) of wild type, P102L, A117V, M129V,
D178N, F198S, E200K and M129V/F198S mutant proteins, respectively.
Sample ready for post
purification analytical techniques
Dialyse against a choice
buffer and store at 4ºC
Check the concentration of the
isolated fractions
Elute bound prions
Load column and wash to clear
non-specifically bound protein
Fusion tag for purification
Sites of Glycosylation. Residues 181 and 197
Figure 1. Protein sequence and expression plasmid map. Residues
90-231 of SHPrP were fused to the His-6 tag of pET-15b for expression
and subsequent purification. Protein landmarks are outlined according to
information from previous structural studies (2). The visual
representation of the construct was created using Plasmapper (3).
Status
sh wild type
Purified
45
4
50 mM Na2PO4,
pH 8.0
sh P102L
Purified
41
4.1
100 mM K2PO4,
pH 8.0
sh A117V
Purified
21
3
10 mM Tris, 100
mM K2PO4, pH 8.0
sh M129V
Purified
35
3
10 mM Tris, 100
mM K2PO4, pH 8.0
sh D178N
Purified
32
4
10-50 mM NaOAc,
pH 4.0-5.0
sh F198S
Purified
35
4
20 mM Tris, 100
mM NaCl, pH 8.0
sh E200K
Purified
30
4
10-50 mM NaOAc,
pH 4.0-5.5
sh M129V
/F198S
Expressed
In progress In progress
In progress
mm wild type
Expressed
In progress In progress
In progress
Solubility
(mg/ml)
Buffer
Condition
15
Centrifuge lysate to clear
the sample for column loading
Cysteines involved in disulfide bonds Residues 179 and 214
Recombinant
PrPc(90-231)
Yield
(mg/L of
culture)
15
20
Harvest culture and lyse
using freeze/thaw method
in denaturing buffer
Beta sheets
kDa
20
Growth and expression in
M9 media
Alpha Helices
AVAILABLE PRION PROTEIN SAMPLES
SHPrPc (90-231)
PURIFICATION PROTOCOL
90
MGSSHHHHHHSSGLVPRGSHMLGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAV
VGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNF
VHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS
231
PURIFIED PROTEIN SAMPLES
Use a gradient from denaturing to
non-denaturing conditions to slowly
refold the prion
Figure 3. Nickel-NTA Column Purification. Cells were taken from
frozen glycerol stocks and grown in 100 ml LB with Ampicillin selection
overnight. This starter culture was used to inoculate 500 ml of M9 media
to an O.D. between 0.6 and 1.0. Culture was grown for 1 hour before
induction using IPTG. Post induction culture was left to produce SHPrPc
for 18 hours. Cells were harvested by centrifugation at 3,000 rpm for 25
minutes at 4ºC, resuspended in lysis buffer (8 M Urea, 10 mM Tris, 100
mM K2PO4, 10 mM reduced Glutathione, pH 8.0) and subjected to 5
rounds of freeze/thaw. Lysate was cleared by centrifugation at 12,000
rpm for 1 hour and 10 minutes. Cleared lysate was added to 35 ml of
nickel-NTA resin for sufficient binding time. The column was washed with
100 ml of denaturing buffer (8 M Urea, 10 mM Tris, 100 mM K2PO4, pH
8.0). The proteins were refolded using a 200 ml gradient going from
denaturing buffer to refolding buffer (10 mM Tris, 100 mM K2PO4, pH
8.0). The column was subsequently washed with 200 ml of refolding
buffer and 100 ml of refolding buffer containing 50 mM Imidazole.
Refolded protein was eluted with 200 ml of a competitive nickel-binding
buffer (500 mM Imidazole 10 mM Tris, 100 mM K2PO4, pH. 5.6). 5 ml
fractions were collected and sampled for eluted prions. Samples were
dialysed against appropriate buffers for further concentration. The
protocol was adapted from the method described by Zahn et al. (4).
Figure 4. Coomassie blue stained gel image of purified wild type SHPrPc(90-231).
Small volumes (5-10 µl) of purified protein fractions were analyzed by SDS-PAGE using
a 15% acrylamide gel. Protein bands showed an appropriate size of 18 kDa with high
purity. The purified samples were subsequently quantified by Bradford assay.
OFF-COLUMN REFOLDING
For most mutant proteins, we encountered difficulties
with on-column refolding and elution under native
conditions. Purified mutant proteins were more efficiently
eluted from the Ni-NTA column under denaturing
conditions, and refolded by dialyzing against appropriate
buffers. Yields were decreased due to the last
conditioning step.
Denatured prion protein in
8M Urea or 6M GuHcl
10 volumes of initial refolding buffer
with 1.1 M GuHCl
Dialyze against 20 volumes of
dialysis buffer
Figure 5. Off-column refolding scheme. Denatured protein solution was added dropwisely to initial refolding buffer (1.1 GuHCl, 55 mM Tris, 21 mM NaCl, 0.8 mM KCl, 1 mM
GSH, 1 mM GSSG, pH 8.2). Total volume of the protein solution was dialyzed against an
appropriate buffer using dialysis tubing with 3 kDa cut-off.
DETERMINING OPTIMAL BUFFER CONDITION
Purified and refolded prion protein was concentrated
using an Amicon Ultra centrifugal filter. A small portion of
concentrated sample (10-15 µl) was used to perform
button test with microdialysis cells to investigate the
solubility of the protein in various buffer conditions. The
microdialysis cells were incubated in test buffer solutions
at room temperature for up to 2 weeks.
Table 1. Summary of the available prion protein samples
(sh and mm refer to Syrian Hamster and Mus musculus)
CONCLUSION
We are continuing to optimize the expression,
purification and refolding procedures in order to produce
sufficient amounts of each mutant protein to perform
complete structural characterization. All constructs are
available as BL21(DE3) stab cultures. Purified and
refolded protein samples are also available upon
request.
REFERENCE
(1) Prusiner, S.B. (1996). Molecular biology and pathogenesis of prion diseases. Trends Biochem. Sci. 21, 482-487
(2) James, T.L., Liu, H., Ulyanov, N.B. et al. (1997). Solution structure of a 142-residue recombinant prion protein
corresponding to the infectious fragment of the scrapie isoform. PNAS 94, 10086-10091
(3) Dong, X., Stothard, P., Forsythe, I.J. and Wishart, D.S. (2004) Plasmapper: a web server for drawing and autoannotating plasmid maps. Nucleic Acids Res. 1: 32, 660-4.
(4) Zahn, R., Liu, A., Lührs, T., Riek, R., et al. (2000) NMR solution structure of the human prion protein. PNAS
97:1, 145-150
ACKNOWLEDGEMENTS
We thank our funding sources, PrioNet Canada and Alberta Prion Research Institute and Alberta Ingenuity Fund.