Article Rapid determination of protein solubility and stability conditions for NMR studies using incomplete factorial design Thierry Ducat, Nathalie Declerck, Thierry Gostan, Michel Kochoyan & He´le`ne De´me´ne´* A contribution from the Centre de Biochimie Structurale, CNRS UMR 5048/INSERM UMR 554/Universito de Montpellier 1, 29 rue de Navacelles, 34090, Montpellier Cedex, France Received 1 August 2005; Accepted 23 December 2005 Key words: incomplete factorial design, sample preparation, solubility, stability Abstract Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample condi- tions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters. Abbreviations: BME – beta-mercaptoethanol; DTT – dithiothreitol; EDTA – ethylenediamine tetraacetic acid; FFD – full factorial design; IFD – incomplete factorial design; IPTG – isopropyl-beta-D-thiogalac- topyranoside; LB – Luria Bertani; SAS – solubility and stability; SDS-PAGE – sodium dodecyl sulfate- polyacrylamide gel electrophoresis; TRIS – tris-(hydroxymethyl) aminomethane. Introduction Remarkable progress has been achieved in recent years for the acquisition and use of NMR data (Pervushin et al., 1997; Tjandra and Bax, 1997; Frydman et al., 2002; Kim and Szyperski, 2003; Kupce and Freeman, 2003). Nevertheless, the lim- iting step in structural studies of macromolecules often remains the preparation of samples suitable for experimental analysis. The determination of protein structures by NMR spectroscopy still often requires samples that are soluble and stable for several days at millimolar concentrations, at pref- erably acidic pH and temperature above 30 °C. For this purpose, the determination of protein solubility and stability (SAS) conditions is crucial, yet it remains mostly empirical and hence time- and material-consuming. Very few studies have been conducted in order to develop rapid and general *To whom correspondence should be addressed. E-mail: helene.demene@cbs.cnrs.fr Journal of Biomolecular NMR (2006) 34: 137–151 Ó Springer 2006 DOI 10.1007/s10858-006-0003-0