The chapter describes several common procedures for use of cell-free expression systems, including strategies to obtain homogeneous populations of nascent polypeptides, separation of ribosomal complexes, induced release of nascent polypeptides from ribosomes, and immunoadsorption of synthesized polypeptides. Factors as compactness, structural stability, cooperativity of folding, and some local structural properties of polypeptides can be studied by applying such techniques as size-exclusion chromatography under native and denaturing conditions, urea-gradient electrophoretic analysis, and limited proteolysis. The chapter presents an approach to study the kinetic mechanism of biosynthetic protein folding and assembly. The chapter also discusses approaches to evaluate in a complex mixture the equilibrium binding affinity of specific proteins. The method that is related to the enzyme-linked immunosorbent assay (ELISA) competitive binding technique, allows analysis of protein-protein interactions and, more generally, protein-ligand interactions in complex mixtures even when the interacting components are present at low levels, as is the case in cell-free expression systems. The physical and chemical principles that allow detailed descriptions of molecular interactions in comparatively simple reactions also control reactions under the complex conditions of cell-free protein synthesis. Series: Methods in Enzymology (Book 290) Hardcover: 500 pages Publisher: Academic Press; 1 edition (March 19, 1998) Language: English ISBN-10: 0121821919 ISBN-13: 978-0121821913 Product Dimensions: 6 x 1.2 x 9 inches Link Download http://nitroflare.com/view/2B04F77D2170923https://drive.google.com/drive/folders/1yLBzZ1rSQoNjmWeJTZ3WGQHg04L1
