Extraction and Purification of Proteins from E. Coli without Harvesting Cells

. Extraction and separation of foreign proteins expressed in E. coli commonly employs multistep fractionation procedures combining centrifugation, mechanical disruption, volume reduction, filtration, and finally capture and recovery in order to separate cells from culture media and purify the target protein from the cell extracts[1,2]. These methods are not amenable to rapid high throughput (HT) purification of thousands of proteins. We demonstrate here a HT-compatible reagent, PopCulture™, and associated method that eliminate the necessity for multiple mechanical fractionation procedures. The entire culturing, extraction, and purification can be achieved in the original culture tube or multiwell plate. This "in-media" protein purification procedure may be adapted to high throughput robotic processing of samples for proteomics research and any application that would benefit from the increased speed and convenience it provides. METHOD. Protein is isolated by culturing cells in liquid media under conditions for optimal target protein expression, chemically lysing the cells directly in their liquid media with a concentrated mixture of specialized detergents and buffer (*PopCulture™ Reagent), reduction of the extract’s viscosity by the addition of *Benzonase® nuclease and *lysozyme, capturing the target protein by adsorption to an *affinity matrix, washing the capture matrix-target protein complex to remove spent culture media and unwanted cellular contaminants, and elution of the purified protein from the adsorptive matrix (*Novagen Inc., Madison, WI). RESULTS. We have employed the PopCulture Reagent and protocol to purify a GST Tag™/His Tag® fusion protein using immobilized glutathione (GST-Bind Resins) or immobilized metal chelation (IMAC; His-Bind® Resins). The results are summarized in Table 1 and Figs. 1 and 2.