Chapter 52 : Purification and Characterization of Proteins

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This chapter provides an introduction to the fascinating, useful, and sometimes dreaded world of protein purification and characterization. The goals of purification vary with the intended use of the purified protein. Protein purification can generally be divided into five broad stages, which do not all need to occur sequentially: (i) preparation of the source, (ii) gathering of all available information about the protein’s properties, (iii) development of an assay, (iv) primary isolation, and (v) final purification. In recent years an autoinduction medium originally developed by Studier and now distributed by EMD Chemicals has gained an increased following in the protein expression community. Recovery and initial isolation steps separate the product from the majority of the water in the cultivation medium and from the majority of the host cell components. Recovery of both extracellular and intracellular proteins thus usually begins with the separation of the soluble culture medium from the insoluble cell pellet by centrifugation or filtration. Ion-exchange chromatography is the most common high-resolution method for preparative separation of proteins and is used in most protocols. Many eukaryotic (and a few prokaryotic) proteins contain sugars, which are sometimes essential to the protein’s function. For instance, we now know that glycosylated proteins are involved in a wide variety of crucial molecular recognition events. Available methods include stains and blots for detection of glycosylation, deglycosylating enzymes, sugar-specific lectins for blotting and separations, and nuclear magnetic resonance and mass spectrometry (MS) methods of characterizing individual side chains.

Citation: Strych U, Willson R. 2010. Purification and Characterization of Proteins, p 731-742. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch52

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Overview of a typical protein purification protocol. After being expressed in the host, the protein of interest will be found in the supernatant or will remain inside the cells, either soluble or as inclusion bodies. HIC, hydrophobic interaction chromatography; IEX, ion-exchange chromatography.

Citation: Strych U, Willson R. 2010. Purification and Characterization of Proteins, p 731-742. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch52
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Types of liquid chromatography

Citation: Strych U, Willson R. 2010. Purification and Characterization of Proteins, p 731-742. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch52
Generic image for table

Modes of adsorptive chromatography

Citation: Strych U, Willson R. 2010. Purification and Characterization of Proteins, p 731-742. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch52
Generic image for table

Affinity chromatography using protein tags

Citation: Strych U, Willson R. 2010. Purification and Characterization of Proteins, p 731-742. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch52

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