1. Construction of Gateway TM -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 Design of Gateway TM -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. 1 Note: GST: glutathione S-transferase, His6: 6 histidine tag, MBP: Maltose binding protein, NusA: N-utilizing substance A, TRX: thioredoxin. T7 Prolac opRBS KpnI Gateway cassetteC-ter His6T7 Term. pGWA ATG Abstract. We describe the construction of a 10 Gateway TM -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 Gateway TM -based compatible vector set for co-expressing multi-protein complexes directly in E. coli. Amp r pBR322 ori lacI p0GWANone pGGWA GST pMGWA MBP pNGWA NusA pXGWA TRX pHGWA His6 pHGGWA His6GST pHMGWA His6MBP pHNGWA His6NusA pHXGWA His6TRX Validation of Gateway TM -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 Gateway TM -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. 2 3 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. ORF-His6 GST-ORF-His6 MBP-ORF-His6 NusA-ORF-His6 TRX-ORF-His6 His6-ORF His6-GST-ORF His6-MBP-ORF His6-NusA-ORF His6-TRX-ORF E0508 E0511 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. - 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. => Tag’s nature does not impair His6 tag accessibility whatever its location. => 4 Design of Gateway TM -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. pCoGWApET-DuetColE1Ampicillin pCoGWSpCDF-Duet CDF Spectinomycin pCoGWCpACYC-Duetp15AChloramphenicol pCoGWKpCOLA-DuetCOLAKanamycin 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). VectorBased onOriginResistance Busso, D. et al. (2005) Anal. Biochem. (in press)