Protein Biochemistry

Protein purification involves a series of processes intended to isolate one or a few proteins from a complex mixture, such as lysates of prokaryotic and eukaryotic cells, or even tissues. Protein purification is vital for the characterization of the function, structure and interactions of proteins of interest. The purification process serves to separate the desired protein from all other proteins. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity [1]. There are many different expression systems, such as bacteria, yeast, plants and insect cells used for the production of proteins of interest. The CTB group mainly uses bacterial and insect cell expression systems for the production of the proteins used in their research. The Auer group uses the ÄKTAexplorer 100 FPLC (Fast Protein Liquid Chromatography) system for the purification of recombinant proteins via affinity tag (His-tag, GST-tag, StreptagII®), anion and cation exchange and size exclusion chromatography. Quality and purity of the purified proteins are verified by SDS-PAGE gel electrophoresis and HPLC (High-Performance Liquid Chromatography) on reversed-phase and analytical size exclusion columns. Purified proteins are used unlabelled, or after fluorescent labelling and/or chemical tagging in one of CTBs 3 experimental assay and screening platforms, LFAP (Label Free Affinity Screening), CONA (Bead based screening) or Fluorescence based screening using ensemble average or single molecule regimes.

[1]. Protein production and purification. Nature Methods, 2008, 5:2, 135-146.