Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients

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 interrogation 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.

Download Full-text

Complexome Profiling: Assembly and Remodeling of Protein Complexes

International Journal of Molecular Sciences ◽

10.3390/ijms22157809 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7809

Author(s):

Ilka Wittig ◽

Pedro Felipe Malacarne

Keyword(s):

Mass Spectrometry ◽

Density Gradient Centrifugation ◽

Protein Complexes ◽

Gradient Centrifugation ◽

Protein Assemblies ◽

Quantitative Mass Spectrometry ◽

Macromolecular Complexes ◽

A Cell ◽

Health And Disease ◽

Native Gel

Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.

Download Full-text

Cesium sulfate density gradient centrifugation of biological macromolecules in the presence of aprotic polar solvents

Journal of Polymer Science Part C Polymer Symposia ◽

10.1002/polc.5070390126 ◽

2007 ◽

Vol 39 (1) ◽

pp. 293-304

Author(s):

C. W. Hahn

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Biological Macromolecules ◽

Polar Solvents ◽

Cesium Sulfate

Download Full-text

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100064037 ◽

1969 ◽

Vol 27 ◽

pp. 424-425

Author(s):

Lee F. Ellis ◽

Richard M. Van Frank ◽

Walter J. Kleinschmidt

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Uranyl Acetate ◽

Interferon Inducer ◽

Virus Particles ◽

Staining Methods ◽

Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

Download Full-text

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665326 ◽

1983 ◽

Vol 50 (04) ◽

pp. 848-851 ◽

Cited By ~ 11

Author(s):

Marjorie B Zucker ◽

David Varon ◽

Nicholas C Masiello ◽

Simon Karpatkin

Keyword(s):

Density Gradient ◽

Combining Ability ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Electrophoretic Pattern ◽

Sucrose Density Gradient ◽

Irreversible Loss ◽

Sucrose Density Gradient Centrifugation ◽

Crossed Immunoelectrophoresis ◽

Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Download Full-text