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Expression of eukaryotic genes in the cytoplasm of Escherichia coli often leads to the production and accumulation of the recombinant protein (r-protein) to levels of total cell protein that are higher than when secreted, in some instances by as much as 25%. The r-proteins so produced are frequently in the form of insoluble aggregates, so-called inclusion bodies (IBs). The IBs contain reduced and denatured proteins lacking biological activity. Their sizes and densities are also distinct from most of the bacterial proteins, which facilitates their separation from intracellular E. coli proteins and cell debris by slow speed centrifugation. A general protocol for the extraction and purification of IBs from E. coli cells is shown. The purified IBs are usually dissolved in 6 M guanidine hydrochloride or 8 M urea at near neutral pH prior to re-naturation and purification of the r-protein. The most commonly used procedure for refolding of such de-natured r-proteins is slow dialysis, or dilution into a buffer of near neutral pH. This result in a significant dilution of the r-protein, typically to a few mg of r-protein per mL of solution, with the formation of precipitated proteins being often unavoidable. Alternative purification and/or refolding procedures using gel filtration, ion exchange or hydrophobic interaction chromatography (HIC) have been developed to circumvent such problems. These are outlined briefly in this Application Note. Close

This handbook describes the use of SEC for the purification and separation of biomolecules, with a focus on practical information for obtaining the best results. The media available, selection criteria and examples with detailed instructions for the most common applications are included, as well as the theoretical principles behind the technique. The first step towards a successful separation is to select the correct medium and this handbook focuses on the most up-to-date SEC media and prepacked columns. Close

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