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Protein Purification Flow Charts

R.K. Scopes¹

¹La Trobe University, Bundoora, Australia

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 1.3

DOI:

10.1002/0471140864.ps0103s00

Online Posting Date:

May, 2001

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Abstract

Protein purification flow charts are presented to give a broad outline of the methods used for different types of proteins. They cannot give any detail, as the process appropriate for each protein will have its own variations at each stage. In most cases, the first stage is to obtain a solution containing the desired protein, after which it can be fractionated by various separation techniques. This unit provides charts tailored for purification of soluble and insoluble recombinant and non-recombinant proteins, as well as membrane-associated proteins.

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Unit Introduction
Soluble Recombinant Proteins
Insoluble Recombinant Proteins
Soluble Nonrecombinant Proteins
Membrane-Associated and Insoluble Nonrecombinant Proteins
Bibliography
Figures

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Figures

Figure 1.3.1

Purification scheme for soluble recombinant proteins, which may be excreted or located in the periplasm, in the membrane fraction, or most commonly the cytoplasm. The first step is to obtain an extract containing the desired protein in soluble form. After this, conventional purification steps may be carried out, or affinity purification of tagged fused proteins can be performed.

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Figure 1.3.2

Purification scheme for insoluble recombinant proteins that are produced as inclusion bodies in the cytoplasm of host cells. The cells must be broken open, and then the insoluble inclusion bodies are separated by differential centrifugation. Solubilization is achieved by the use of denaturing solvents, and renaturation of the dissolved protein occurs on removal of the denaturant. Further polishing steps will be needed to remove small amounts of contaminating proteins as well as incorrectly folded species. Additional information can be found in unit 6.3-6.5.

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Figure 1.3.3

Purification scheme for soluble proteins present in their natural host cells. Cells must be disrupted to release the proteins, usually in the presence of 2 to 10 ml of a suitable buffer per gram weight. After removal of insoluble material, the process will generally require several steps, using various standard fractionation procedures in a suitable order. For production of highly pure protein, a final polishing step may be required to remove final trace contaminants. Additional information can be found in unit 6.2.

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Figure 1.3.4

Purification scheme for membrane-associated and poorly soluble proteins (nonrecombinant). An initial purification can be achieved by isolation of organelles containing the desired protein. Membrane proteins are normally solubilized with a nonionic detergent, although loosely associated proteins may be extracted without detergent at high pH,with EDTA, or with small amounts of an organic solvent such as N-butanol. Normal fractionation procedures may need some modification if the detergent is required throughout to maintain the integrity of the protein.

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Literature Cited

Literature Cited
	Walter, H. and Johansson, G. (eds.) 1994. Aqueous two-phase systems. Methods Enzymol. 228:1-725.
	Zeilstra-Ryalls, J., Fayet, O., and Georgopoulos, C. 1991. The universally-conserved GroE (Hsp60) chaperonins. Annu. Rev. Microbiol. 45:301-325.
Key References
	Deutscher, M.P.(ed.) 1990. Guide to protein purification. Methods Enzymol. 182:1-894.
Extensive collection of purification methods, with some general protocols and examples.
	Janson, J.-C. and Ryden, L.G. 1989. Protein Purification: Principles, High Resolution Methods, and Applications. VCH Publishers, New York.
A useful collection of methods and examples.
	Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recovery Processes for Biological Materials. John Wiley & Sons, New York.
A useful introduction to the problems of large-scale methods.
	Kenny, A. and Fowell, S. (eds.) 1992. Practical protein chromatography. Methods Mol. Biol. 11:1-327.
Extensive descriptions of affinity chromatographic techniques with protocols and recipes.
	Scopes, R.K. 1993. Protein Purification, Principles and Practice, 3rd ed. Springer-Verlag, New York and Heidelberg.
General principles of all the main techniques used in purifying proteins. A useful laboratory handbook; does not include recipes or procedures for specific proteins.