Transcript Document

Rapid production of fusion
proteins using
the RTS 100/500 Systems
Jan Stracke, Michael Schräml, Andreas Junger, Dorothee Ambrosius and
Martin Lanzendörfer
Roche Diagnostics GmbH, Pharmaceutical Research, Dept. of Biochemistry, Penzberg, Germany
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High need for purified proteins in pharmaceutical research
for:
X-ray crystallography (X-ray)
Nuclear Magnetic Resonance (NMR)
High Throughput Screening (HTS)
Therapeutic Proteins (e.g. antibodies)
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Protein Production
Gene
Protein
Screening of various
constructs for
expression
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Crystal/
Structure
Screening of various
muteins and/or
truncated constructs
for crystallisation
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Bottlenecks of protein crystallisation
cloning
expression
cell lysis
soluble / insoluble protein
refolding
time consuming step
fermentation
purification
characterisation
activity assays, interaction analysis
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high need for acceleration
of protein production
high need for acceleration
of methods
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Classical Protein Production
transcription
gene
&
translation
RTS 500
gene
RTS 100
DNA-template
RTS 100
50 µg / ml
T7-RNA polymerase
E.coli lysate
mRNA
RTS 500
500 µg / ml
protein
RTS 500 HY
5 mg / ml
control of expression by SDS-PAGE, Western blotting, ELISA
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Rapid Translation System (RTS)
Protein
MW
Organism
Function
Yield RTS 500 [µg]
Endostatin
20
human
Inhibitor
100
Erythropoietin
30
human
hormone
550
Interleukin-2
15.4
human
hormone
600
p40phox SH3 mod.
47
human
regulatory protein
90
Phosphodiesterase
56
human
regulatory protein
100
Rab 5
22
human
GTPase
100
41
VEGF-receptor
Green fluorescent p. 28
human
receptor fragment
250
animal
fluorescent protein
560
b -Galactosidase
118
bacteria
enzyme
105
HIV Tat
10
virus
transcription factor
n. d.
s-Adenosylmethionine synthetase 1
41.6
yeast
enzyme
6
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Proteins successfully expressed with RTS
RTS 500
vs
RTS 500 HY
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Successfully expressed proteins - a system comparison
Expression of...
...cell toxic proteins…
...two proteins in parallel...
…in the RTS 500.
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Further applications of RTS
Crystals of phosphoserine phosphatase (PSP)
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Successfully crystallised proteins
The protein was expressed using the RTS 500 E. coli HY Kit. Approximately 3 mg of PSP protein,
fused at the C-terminus with a His6 tag, was produced in a single 1 ml reaction, purified,
concentrated and subsequently crystallised at 25°C using the hanging-drop method.
Data courtesy of Ho S. Cho, Weiru Wang, Sung-Hou Kim and David E. Wemmer, Berkeley Structural
Genomic Center.
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• in vitro batch expression in 50 µl reactions
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RTS 100 expression
• designed for the use of linear PCR-generated DNA templates
• enables the fast and parallel expression and evaluation of
numerous constructs (e.g. for crystal engeneering)
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Schematic illustration of template generation for RTS 100 expression
using PCR
promotor module
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terminator module
cs tag T7T
T7P RBS tag cs
gene X
PCR
T7P RBS tag cs
gene X
cs tag T7T
linear template for in vitro translation (batch)
Various promotor and terminator modules can be prepared and fused by
PCR in numerous combinations with the gene of interest. Expression of
these constructs is performed in the cell-free translation system RTS 100.
MTP format is possible!
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RTS 100 expression
RTS 100 HY vs RTS 500 HY
lower yields in the RTS 100 compared to RTS 500
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Successfully expressed proteins - a system comparison
RTS (in vitro expression)
soluble / insoluble protein
refolding
high affinity purification
activity assays, interaction analysis
goal
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MTP-96 well-format
PCR product (e.g. RT-PCR)
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production in days (not months)
high throughput
high flexibility
automation
small scale production
need for high yield expression
classical or in vitro optimisation
acceleration of protein production
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High Throughput Protein Production (HTPP) using RTS
100
Evaluation of:
•Solubility
•Affinity-tags (purification, immobilisation)
•Ligand activity (BIAcore)
using the RTS 500 system for construct expression.
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Pharmaceuticals
Example 1
MMP2-PEX: hemopexin-like domain of MMP2
MW = 23 kDa; 1 disulfide bridge
TIMP2: tissue type inhibitor of matrixmetallo-proteinase 2
MW = 22 kDa; 6 disulfide bridges
TIMP2 4
1
PEX (MMP2)
4
1
N
3
2
C
3
Williamson et al. (1994)
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2
Gohlke et al. (1996)
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Example 1 - Structures of MMP2-PEX and TIMP2
promotor
E.coli
RTS-500
and
E.coli
fused genes
vector
tac
PinPoint-tag
PEX2
Xa3-PinPoint
tac
PinPoint-tag
TIMP2
Xa3-PinPoint
tac
PinPoint-tag
Protein A
Xa3-PinPoint
T7
AVITAG
PEX2
pIVEX 2.1
AVITAG
pIVEX 2.1
T7
T7
T7
Protein B
Strep-tag
Poly-Glu--tag
PEX2
pIVEX 2.2b Nde
PEX2
pIVEX 2.3 MCS
4 protein coding genes in 7 constructs
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Example 1 - Evaluation of constructs for BIAcore analysis
Relative yields of expressed protein - Improvement of solubility
in the RTS-500 System
E.coli
RTS-500 System
0%
Protein A NtPP
Protein B
NtAT
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Example 1 - Evaluation of constructs for BIAcore analysis
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Analysis of ligand interactions of RTS-expressed PEX2-NtAT
(N-terminaler AVITAG) with TIMP-2 using BIAcore
80
Dissociation
Association
70
TIMP-2 concentration
Response Difference [RU]
60
50
40
30
20
10
Ligand: PEX2_NtAT
Analyte : TIMP2
0
Injection
-10
-50
0
50
100
150
200
250
300
Time [s]
Equilibrium constant KD = 1.5x 10-10M
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350
400
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Example 1 - Evaluation of constructs for BIAcore analysis
Aim: Production of sufficient quantities of active Protein B
for biochemical characterisation and crystallisation.
Evidence exists that:
• a truncated form may be active
or
• co-factors may be required for activity
These aspects are being addressed using the RTS 100
and 500 systems for construct expression/evaluation.
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Example 2 - Production of active Protein B
Co-expression
Truncations/mutations
truncated/mutated constructs by PCR
target protein expression RT-PCR
(RTS 500)
immobilisation
expression in 96 well plates
(RTS 100)
ligand fishing
activity assay
co-expression with
selected
activating proteins
(e.g. RTS 100)
ligand identification
activity assay
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Active
Protein B
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Example 2 - Production of active Protein B
Truncated Protein B constructs, generated by PCR
for expression in the RTS 100
Prot. B
T7P RBS tag cs
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gene B
cs tag T7T
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Example 2 - Production of active Protein B
Aim: Evaluation of approx. 50 muteins of Protein C
(extracellular,
7 disulphide bonds) for crystallisation.
Strategy:
generation of numerous mutated linear constructs by PCR
expression in 96 well plates (RTS 100)
refolding and purification in 96 well plates
assay for ligand activity (e.g. BIAcore)
selection of active and inactive constructs for
large scale expression and
crystallisation
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info about ligand binding site
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Example 3 - Production of various muteins
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Conclusions
• proteins of interest can be expressed in an active, soluble form in the RTS systems
in
µg/mg quantities for initial characterisation (e.g. activity)
• proteins can easily be co-expressed if necessary
• due to the possibility to use linear PCR products as templates, the RTS 100 allows
rapid
parallel screening of numerous protein constructs with regard to activity, ligand
binding
and solubility
the bottleneck
of protein
production
for
• RTS 100Overcoming
is predestinated
for automation
of protein
expression
and purification
crystallography seems possible in the near future using RTS!
J. Stracke, 09/01
Roche Pharma Research
A. Grossmann
R. Engh
M. Dangl
P. Rüger
K. Lang
Max Planck Inst.f.
Biochemie
R. Engh
R. Huber
N. Heim
M. Wisniewska
TU-München
Roche Mol. Biochemicals
J. Buchner
C. Nemetz
A. Gräntzdörfer
S. Wessner
M. Watzle
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Acknowledgements