Construction of Gateway TM -based vectors for high-throughput cloning and (co-)expression screening in Escherichia coli

1. Construction of GatewayTM-based vectors for high-throughput cloning and (co-)expression screening in Escherichia coli pCoGWA pET-Duet ColE1 Ampicillin CDF pCoGWS pCDF-Duet Spectinomycin pCoGWC pACYC-Duet p15A Chloramphenicol pCoGWK pCOLA-Duet COLA Kanamycin 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 20°C. 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 NusA-ORF-His6 His6-NusA-ORF MBP-ORF-His6 His6-MBP-ORF TRX-ORF-His6 His6-TRX-ORF GST-ORF-His6 His6-GST-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) • Tag’s nature and location do not change expression profile. • Larger tags (MBP, NusA) help to solubilize proteins. • His tag location may interfere on protein solubility. => 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 Tag’s nature does not impair His6 tag accessibility whatever its location. => 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).