Pr

1
Construction of GatewayTM-based vectors for
high-throughput cloning and (co-)expression
screening in Escherichia coli
Didier Busso, Loubna Salim, Edouard Troesch,
Bénédicte Delagoutte-Busso, Jean-Claude Thierry
and Dino Moras
Structural Biology and Genomics Department (CNRS
UMR 7104), IGBMC, 1 rue Laurent Fries, Illkirch
67404, France
Abstract. We describe the construction of a 10
GatewayTM-based vector set applicable for
high-throughput cloning and for expressing
recombinant proteins in Escherichia coli.
Plasmids bear elements required to produce
recombinant proteins under the T7 promoter
control and encode different N- terminal
fusion(s). A sequence encoding a 6 histidine tag
has been inserted to be in frame with the cloned
Open Reading Frame (ORF) either at its N-terminus
or at the C-terminus, giving the flexibility of
choosing the 6 histidine tag location for further
purification. Since the vector set is derived
from a unique backbone, a consistent comparison
of the impact of fusion partner(s) on protein
expression and solubility is easily amenable.
Moreover, the presence of the T7 promoter
facilitates parallel expression screening using
auto-inducible media (Studier, F.W. (2005) Prot.
Expr. Purif. 41 207-234). Following the same
strategy, we are now constructing a new
GatewayTM-based compatible vector set for
co-expressing multi-protein complexes directly in
E. coli.
Design of GatewayTM-based expression vectors. All
vectors are derived from the pET22b vector (Merck
- Novagen). The plasmid has been modified in
order to insert the Gateway cassette in frame
with N-terminal fusion(s) and with the C-terminal
6 histidine tag.
Validation of GatewayTM-based expression
vectors. Two ORFs (SBGP code E0508 and E0511)
were amplified by PCR in order to generate two
products attB1-ORF-attB2 and attB1-ORF-STOP-attB2
. The ORFs were sub-cloned within the panel of
GatewayTM-based expression vectors (see 1) and
tested for expression into BL21(DE3) cells.
Parallel cultures were done in auto-inducible
medium (ZYM-5052) at 20C. Same amount of
material were loaded onto SDS-PAGE.
1
2
p0GWA
None
Note GST glutathione S-transferase, His6 6
histidine tag, MBP Maltose binding protein,
NusA N-utilizing substance A, TRX thioredoxin.
pGGWA
GST
pMGWA
MBP
GST-ORF-His6
MBP-ORF-His6
NusA-ORF-His6
TRX-ORF-His6
His6-GST-ORF
His6-MBP-ORF
His6-NusA-ORF
His6-TRX-ORF
ORF-His6
His6-ORF
pNGWA
NusA
pXGWA
E0508
TRX
pHGWA
His6
pHGGWA
His6
GST
pHMGWA
His6
MBP
pHNGWA
His6
NusA
pHXGWA
His6
TRX
T7 Pro
lac op
RBS
Gateway cassette
C-ter His6
T7 Term.
E0511
ATG
KpnI
lacI
Ampr
pGWA
pBR322 ori
Busso, D. et al. (2005) Anal. Biochem. (in press)

• Tags nature and location do not change
  expression profile.
• Larger tags (MBP, NusA) help to solubilize
  proteins.
• His tag location may interfere on protein
  solubility.

gt
Design of GatewayTM-based co-expression
vectors. We have modified vectors from the Duet
series (Merck Novagen) in order to add the
Gateway cassette in frame with the N-terminal 6
histidine tag and to accomodate a
NdeI-BamHI(BglII) cloning in frame with a
C-terminal Flag tag. Vectors present compatible
replication origin and specific resistance genes.
4
Histidine tag accessibility. Soluble fractions
obtained after cell disruption and centrifugation
were applied onto IMAC resin (50µl) dispensed
onto 96-well filter plate (Whatman). After
extensive washes, bound proteins were eluted.
Samples and buffers run through the resin by
applying vacuum (50 mbar). An aliquot of eluted
fraction (10 µl) was loaded onto SDS-PAGE.
3
Vector
Based on
Origin
Resistance
Tags nature does not impair His6 tag
accessibility whatever its location.
gt
Note GST glutathione S-transferase, His6 6
histidine tag, M molecular weight marker, MBP
Maltose binding protein, NusA N-utilizing
substance A, S soluble proteins, T total
proteins, TRX thioredoxin.
Acknowledgements We thank all members of the
Structural Biology and Genomics Department. This
work was supported by funds from SPINE EEC
QLG2-CT-2002-00988, FNS through the Genopole
program, CNRS, INSERM, ULP and local authorities
(Region of Alsace, Department of Bas-Rhin and
city of Strasbourg).