Developments of protocols for the preparation of glucocorticoid

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 SUMMARY 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 coexpression protocols in E. coli and its limitation are described. GR BIOINFORMATIC ANALYSIS TIF 2 BIOINFORMATIC ANALYSIS A/B Sequences are analyzed using programs developed at the IGBMC: Pipe-Align (http: //titus. ustrasbg. fr/Pipe. Align/), VRP. 1 153 180 214 Goal: find by a high throughput coexpression strategy interacting domains for the co-stabilization of protein complexes C 314 316 D 401 420 E/F specific to NCO 2 PAS domain 486 516 528 777 1 108 183 378 motifs LXXLL 622 Q-rich specific to NCO 2 770 828 1008 1250 1266 1464 GR CONSTRUCTS All the fragments of the human glucocorticoid receptor defined are cloned in the following gateway expression vectors: p. DEST origin resistance construct p 0 GWA Col. E 1 ampicillin GR p. Fla. G Col. E 1 ampicillin Flag-Tb-GR p. HGWA Col. E 1 ampicillin His-Tb-GR p. Et. G 20 A Col. E 1 ampicillin Trx-His-Tb-GR p. Et. G 28 c 2 Col. E 1 kanamycin His-Tb-GR p. Et. G 60 A Col. E 1 ampicillin NUS-His-Tb-GR p. DEST 17 p 15 Azeo p 15 A zeocin His-Tb-GR p. KM 596 G Col. E 1 ampicillin TIF 2 CONSTRUCTS His-MBP-Tb-GR Cell transformation optimization The fragments of TIF 2 including the LXXLL motifs highlighted in red are cloned in the following gateway expression vectors: GR HIS p. Et. G 28 C 2 HIS p. DEST 17 FLAG p. Fla. G NUS p. Et. G 60 A TRX MBP p. Et. G 20 A p. KM 596 G AB 153 -314 ++ ++ ++ AB 153 -401 ++ ++ AB 180 -314 ++ ++ ++ AB 180 -401 ++ ++ + AB 214 -401 ++ ++ ++ ++ CDEF 420 -777 ++ ++ ++ ++ ++ EF 516 -777 ++ ++ ++ Col. E 1 kanamycin His-Tb-TIF 2 p. GGWA Col. E 1 ampicillin GST-Tb-TIF 2 p. MGWA Col. E 1 ampicillin MBP-Tb-TIF 2 Antibiotics: yes / no Temperature: 18°C / 37°C Induction: 0. 4 m. M IPTG / 0. 8 m. M IPTG Type of plate: 24 wells / 96 wells Expression: 1 to 5 hours / o/n ++ ++ - +++ - NUS p. Et. G 60 A ampicillin HIS p. Et. G 28 C 2 kanamycin + +++ HIS p. DEST 17 zeocin +++ ++ NUS (amp) + HIS (zeo) HIS p. Et. G 28 C 2 kanamycin +++ NUS (amp) + HIS (kan) ++ EF 528 -777 - HIS p. DEST 17 zeocin ++ AB 317 -401 + HIS p. HGWA ampicillin ++ ++ AB 214 -314 p. HGWK FRAGMENTS CO-EXPRESSED: GR-Cter X GR-Nter & GR-Cter X TIF 2 ++ ++ construct Results for CO-EXPRESSION LBS/18°C LB/37°C LBS/18°C LBS/18°C ++ resistance Cells: electro-competent / chemically-competent Pre-culture: yes / no Starter for pre-culture: transformed cells / 1 colony Starter for culture: transformed cells / pre-culture / 1 colony Medium: LB / LB+sucrose 10% / auto-induction Results for SINGLE EXPRESSION HIS p. HGWA origin EXPRESSION parameters studied preparation of competent cells cell volume DNA amount of transformed cell used Ca. Cl 2 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 NATIVE p 0 GWA p. DEST DEF 487 -777 TIF 2 ++ HIS p. HGWA GST p. GGWA MBP p. MGWA LBS/18°C 378 -828 + ++ 378 -1008 ++ 622 -770 + ++ + + +++ + NUS +++ MBP (amp) + HIS (zeo) ++ ++ not tested not expressed soluble T S T S AB [153 -314] MBP (amp) + HIS (kan) MBP HIS ++ AB [153 -401] TRX p. Et. G 20 A ampicillin HIS p. DEST 17 zeocin AB [153 -314] EF [516 -777] T S T S ++ 622 -1008 HIS p. Et. G 28 C 2 kanamycin +++ 622 -828 + ++ 378 -1008 378 -828 622 -770 622 -828 622 -1008 T S T S T S ++ HIS p. DEST 17 zeocin T S T S HIS LBS/18°C + MBP p. KM 596 G ampicillin T S T S AB [153 -401] EF [516 -777] +++ + HIS p. Et. G 28 C 2 kanamycin +++ CONCLUSIONS & PERSPECTIVES Single Expression: ● If there is detectable expression level of soluble protein with HIS tag or GST solubilizing fusions (MBP, NUS) are not improving the yield of soluble dispersed production. ● If there is no detectable expression of soluble protein with HIS tag or GST solubilizing fusions like MBP, NUS give good results in term of solubility but the 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 fusion, protein ● 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/TIF 2 complex Proteins and complex purified: AB (153 -314), GR (524 -777) C 638 A, W 557 T, W 712 S / TIF 2 (622 -770) (see poster: Preparation and characterization of complexes between ER and GR nuclear receptor LBDs and TIF 2 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. )) Acknowledgements: We thank all members of the Structural Biology and Genomics Department. This work was supported by funds from SPINE EEC QLG 2 -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).