ABSTRACT
Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12â¯mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.
Subject(s)
Chromatography, Affinity/instrumentation , High-Throughput Screening Assays/instrumentation , Microfilament Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae/chemistry , Chromatography, Affinity/economics , Chromatography, Affinity/methods , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , High-Throughput Screening Assays/economics , High-Throughput Screening Assays/methods , Humans , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Mutation , Pressure , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , VacuumABSTRACT
The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth Ï-value analyses.