1. Single Expression: ● If there is detectable expression level of soluble protein with HIS tag or GST fusion, solubilizing fusions (MBP, NUS) are not improving the yield of soluble dispersed protein production. ● If there is no detectable expression of soluble protein with HIS tag or GST fusion, solubilizing fusions like MBP, NUS give good results in term of solubility but the protein obtained is aggregated. Need strong detergent for dispersion. Co-expression: To maintain a good expression level of the two proteins: ● Important: nature of fusion, antibiotic resistance HIS (kanamycin, zeocin) X NUS, MBP, GST (ampicillin)  OK HIS (kanamycin, zeocin) X HIS, Ø (ampicillin)  not working  Cells: electro-competent / chemically-competent  Antibiotics: yes / no  Pre-culture: yes / no  Temperature: 18°C / 37°C  Starter for pre-culture: transformed cells / 1 colony  Induction: 0.4 mM IPTG / 0.8 mM IPTG  Starter for culture: transformed cells / pre-culture / 1 colony  Type of plate: 24 wells / 96 wells  Medium: LB / LB+sucrose10% / auto-induction  Expression: 1 to 5 hours / o/n The limiting step for structural studies is the ability to obtain large quantities of well characterized functional proteins or complexes. Many important eukaryotic proteins are naturally involved in multi protein complexes and thus are difficult to produce as functional and soluble isolated proteins. Plausible explanations could be unfolding or a high inter domain mobility. Interaction with partner proteins can help to overcome these problems. We describe the definition of domains boundaries using bioinformatics tools, the cloning strategy using the gateway system, the procedure of single expression tests in E. coli microplates using a TECAN robotic station. The co- expression protocols in E. coli and its limitation are described. Developments of protocols for the preparation of glucocorticoid nuclear receptor complexes Eiler, S., Delagoutte, B., Brelivet, Y., Salim, L., Busso, D., Poch, O., Moras, D. and Ruff, M. Structural Biology and Genomics Department (CNRS – UMR 7104), IGBMC, Illkirch 67404, France 1153180214314316401420486516528 777 A / BCDE / F GR BIOINFORMATIC ANALYSIS TIF2 BIOINFORMATIC ANALYSIS specific to NCO2 PAS domainmotifs LXXLL specific to NCO2 Q-rich 1108183378 6227708281008 1250 1266 1464 SUMMARY pDESToriginresistanceconstruct pFlaGColE1ampicillinFlag-Tb-GR pEtG20AColE1ampicillinTrx-His-Tb-GR pEtG60AColE1ampicillinNUS-His-Tb-GR pKM596GColE1ampicillinHis-MBP-Tb-GR GR CONSTRUCTS TIF2 CONSTRUCTS pDESToriginresistanceconstruct pHGWKColE1kanamycinHis-Tb-TIF2 pGGWAColE1ampicillinGST-Tb-TIF2 pMGWAColE1ampicillinMBP-Tb-TIF2  preparation of competent cells  cell volume  DNA amount  amount of transformed cell used  CaCl2 concentration  antibiotic concentration Transformation with two plasmids:  chemically-competent cells need 10 X more DNA than electro-competent cells  efficiency is 100 X less with chemically-competent cells than with electro-competent cells  use of chemically-competent cells  scale-up Cell transformation optimizationEXPRESSION parameters studied GR NATIVE p0GWA HIS pHGWA HIS pEtG28C2 HIS pDEST17 FLAG pFlaG NUS pEtG60A TRX pEtG20A MBP pKM596G LBS/18°C LB/37°CLBS/18°C LB/37°CLBS/18°CLB/37°CLBS/18°C AB 153-314+++ + + AB 153-401++ AB 180-314++ +++ AB 180-401++ + AB 214-314++ + AB 214-401++ + AB 317-401++ + CDEF 420-777+++ + + DEF 487-777++ + EF 516-777+++ + + EF 528-777++ + + TIF2 HIS pHGWA GST pGGWA MBP pMGWA LBS/18°C 378-828++++ 378-1008++ 622-770++++++ 622-828++++ 622-1008++++ Results for SINGLE EXPRESSIONResults for CO-EXPRESSION not tested not expressed ++ expressed ++ soluble HIS pHGWA ampicillin HIS pEtG28C2 kanamycin HIS pDEST17 zeocin +++ - - - TRX pEtG20A ampicillin HIS pEtG28C2 kanamycin HIS pDEST17 zeocin +++ - + NUS pEtG60A ampicillin HIS pEtG28C2 kanamycin HIS pDEST17 zeocin +++ + ++ MBP pKM596G ampicillin HIS pEtG28C2 kanamycin HIS pDEST17 zeocin +++ + + TS 622-770 TSTSTSTS 378-1008378-828622-828622-1008 CONCLUSIONS & PERSPECTIVES TSTSTSTS TSTSTSTS NUS (amp) + HIS (zeo)NUS (amp) + HIS (kan) NUS HIS TSTSTSTSTSTSTSTS MBP (amp) + HIS (zeo)MBP (amp) + HIS (kan) MBP HIS AB [153-314]AB [153-401]EF [516-777] All the fragments of the human glucocorticoid receptor defined are cloned in the following gateway expression vectors: The fragments of TIF2 including the LXXLL motifs highlighted in red are cloned in the following gateway expression vectors: FRAGMENTS CO-EXPRESSED: GR-Cter X GR-Nter & GR-Cter X TIF2 Sequences are analyzed using programs developed at the IGBMC: Pipe-Align (http://titus.u- strasbg.fr/PipeAlign/), VRP.http://titus.u- strasbg.fr/PipeAlign/ Goal: find by a high throughput co- expression strategy interacting domains for the co-stabilization of protein complexes pDESToriginresistanceconstruct p0GWAColE1ampicillinGR pHGWAColE1ampicillinHis-Tb-GR pEtG28c2ColE1kanamycinHis-Tb-GR pDEST17p15Azeop15AzeocinHis-Tb-GR 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). ● Best results without antibiotics ● No effect of origin of replication ● Solubilizing fusion (NUS) is important to maintain a good expression level of GR-EF to produce a stable dispersed GR-EF/TIF2 complex Proteins and complex purified: AB (153-314), GR (524-777) C638A,W557T,W712S / TIF2 (622-770) (see poster: Preparation and characterization of complexes between ER and GR nuclear receptor LBDs and TIF2 domain (Eiler and al.)) Perspectives: Use the bi-cistronic vectors developed in house (see poster: Constructions of gateway-based vectors for high throughput cloning and (co-)expression Screening in E. coli (D. Busso and al.))