Seminars Archive

Protein characterisation by Size Exclusion Chromatography coupled to Static Light Scattering

Abstract
After protein expression has been achieved in sufficient quantities for functional or structural studies, quality control of purified soluble forms is fundamental for ensuring experimental reproducibility. Similarly, the effect of purification conditions (ionic force, pH, sample freezing, …..) on sample aggregation / oligomerisation state is important to estimate the biological relevance of biochemical studies. In this context, Static and Dynamic Light Scattering measurements coupled to Size Exclusion Chromatography is a promising technique, not suffering of usual bias affecting batch measurements. Examples will be exposed on the evaluation of: i) sample homogeneity (mono- / poly-dispersity), ii) oligomerisation state in solution, iii) protein-protein interaction and iv) complex formation with other chemical species, such as detergent micelles and sugar modifications. The possible applications of this technique range from the mere quality control of prepared samples in view of functional characterization, up to the collection of data with some biological relevance. Examples will be provided concerning, in particular, the purification of a membrane Mg++ transporter (CorA homologue) from archaeal species 1, the preparation of a 1000 kDa macromolecular complex (the baseplate from lactococcal phage p2) in view of protein crystallization and the determination of a topological model for the 2000 kDa baseplate from lactococcal phage Tuc2009 2. References 1. Veesler D et al. (2009) Anal Biochem, 388, 115-21. 2. Sciara G et al. (2007) JBC, 283, 2716-23.