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protein purification; PEG precipitation; protein precipitation; chromatography-free protein purification; sucrose density gradient centrifugation; 20S proteasome; 26S proteasome; fatty acid synthase; immunoproteasome; buffer optimization; monolithic columns; macromolecular complexes
Abstract:
A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to
the development of human diseases. Furthermore, detailed mechanistic nterrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however,
chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.
