title: Overview of the purification of recombinant proteins.
abstract: When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same.
authors.affiliation: Bethesda, Maryland.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wingfield
mesh_terms: D000345:Affinity Labels; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D011994:Recombinant Proteins
keywords: protein expression; protein folding; protein purification; protein purification strategies; recombinant protein
references.citation: Methods Enzymol. 1985;114:49-77
references.pmid: 4079776
references.citation: Mol Biol Cell. 1994 Mar;5(3):253-65
references.pmid: 8049518
references.citation: Curr Opin Struct Biol. 2000 Feb;10(1):26-33
references.pmid: 10679467
references.citation: Int J Biochem. 1979;10(11):877-83
references.pmid: 389690
references.citation: J Struct Biol. 1999 Jun 15;126(2):131-44
references.pmid: 10388624
references.citation: J Bacteriol. 1987 Feb;169(2):751-7
references.pmid: 3027045
references.citation: J Biol Chem. 1994 Aug 5;269(31):19910-5
references.pmid: 7914193
references.citation: FEBS Lett. 1986 Sep 15;205(2):200-4
references.pmid: 3091401
references.citation: Methods Enzymol. 1984;107:305-29
references.pmid: 6503716
references.citation: Science. 1991 Dec 20;254(5039):1779-82
references.pmid: 1837174
references.citation: J Bacteriol. 1987 Sep;169(9):4124-7
references.pmid: 3305480
references.citation: Microbiol Rev. 1996 Sep;60(3):512-38
references.pmid: 8840785
references.citation: Curr Opin Biotechnol. 2001 Apr;12(2):208-11
references.pmid: 11287239
references.citation: Methods Enzymol. 1999;309:217-36
references.pmid: 10507027
references.citation: Biochem J. 1997 Sep 15;326 ( Pt 3):763-72
references.pmid: 9307026
references.citation: Curr Opin Struct Biol. 2013 Jun;23(3):345-56
references.pmid: 23623336
references.citation: Biochemistry. 1991 Mar 19;30(11):2790-7
references.pmid: 2007117
references.citation: Methods Enzymol. 1999;309:605-32
references.pmid: 10507051
references.citation: Biochim Biophys Acta. 2000 Nov 23;1508(1-2):86-111
references.pmid: 11090820
references.citation: Trends Biotechnol. 2006 Apr;24(4):179-85
references.pmid: 16503059
references.citation: Methods Enzymol. 1994;239:349-63
references.pmid: 7830590
references.citation: Trends Biochem Sci. 1991 Oct;16(10):358-62
references.pmid: 1785135
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1171-84
references.pmid: 21924345
references.citation: Protein Expr Purif. 2005 May;41(1):98-105
references.pmid: 15802226
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1100-1
references.pmid: 22227398
references.citation: Protein Expr Purif. 1993 Oct;4(5):465-72
references.pmid: 7504538
references.citation: Biotechnology (N Y). 1992 Sep;10(9):1013-9
references.pmid: 1368998
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13703-8
references.pmid: 10570136
references.citation: Nat Struct Biol. 1998 Jul;5 Suppl:496-9
references.pmid: 9665177
references.citation: J Biosci Bioeng. 2005 Apr;99(4):303-10
references.pmid: 16233795
references.citation: Cell. 1985 Jun;41(2):587-95
references.pmid: 3886165
references.citation: Methods Enzymol. 1995;251:8-28
references.pmid: 7651233
references.citation: Biotechniques. 2001 Aug;31(2):322-3, 326-8
references.pmid: 11515368
references.citation: Eur J Biochem. 1987 Jun 15;165(3):537-41
references.pmid: 3297693
references.citation: Curr Opin Biotechnol. 2006 Aug;17(4):353-8
references.pmid: 16781139
references.citation: Biomolecules. 2014 Feb 20;4(1):235-51
references.pmid: 24970214
references.citation: Biochemistry. 1989 Feb 21;28(4):1884-91
references.pmid: 2497771
references.citation: Eur J Biochem. 1974 Sep 1;47(2):409-15
references.pmid: 4607014
references.citation: Methods Enzymol. 1990;185:187-95
references.pmid: 2199779
references.citation: Methods Enzymol. 1990;182:132-43
references.pmid: 2179663
references.citation: Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119-23
references.pmid: 2164223
references.citation: J Virol. 1997 Dec;71(12):8955-61
references.pmid: 9371551
references.citation: J Biol Chem. 1999 Jul 23;274(30):21362-8
references.pmid: 10409697
references.citation: Protein Sci. 1997 Jun;6(6):1248-63
references.pmid: 9194185
references.citation: Annu Rev Biochem. 1978;47:481-532
references.pmid: 354502
references.citation: Biochemistry. 2005 Dec 13;44(49):16167-80
references.pmid: 16331977
references.citation: Protein Eng. 1994 Jul;7(7):933-9
references.pmid: 7971955
references.citation: Anal Biochem. 1995 Sep 1;230(1):8-15
references.pmid: 8585634
references.citation: Biotechnology (N Y). 1994 Dec;12(13):1357-60
references.pmid: 7765566
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1185-94
references.pmid: 22008973
references.citation: Gene. 1990 Mar 15;87(2):243-8
references.pmid: 2158928
references.citation: Biochemistry. 1993 Mar 16;32(10):2469-72
references.pmid: 8448106
references.citation: Biotechnol Genet Eng Rev. 2012;28:147-75
references.pmid: 22616486
references.citation: Biotechnology (N Y). 1991 Sep;9(9):825-9
references.pmid: 1367356
references.citation: EMBO J. 1998 Aug 17;17(16):4572-84
references.pmid: 9707417
references.citation: Protein Sci. 2004 Jul;13(7):1802-10
references.pmid: 15215523
references.citation: PLoS One. 2011;6(7):e22981
references.pmid: 21829569
references.citation: Appl Biochem Biotechnol. 1992 May;33(2):117-38
references.pmid: 1380790
references.citation: Methods Enzymol. 1984;107:301-4
references.pmid: 6503715
references.citation: Microb Cell Fact. 2004 Sep 2;3(1):11
references.pmid: 15345063
references.citation: FEBS J. 2012 Jul;279(13):2272-82
references.pmid: 22594874
references.citation: Science. 1983 Feb 11;219(4585):660-6
references.pmid: 6186024
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1158-70
references.pmid: 21968146
references.citation: Nature. 1990 Apr 19;344(6268):715-6
references.pmid: 1691821
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5810-4
references.pmid: 20231488
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):422-7
references.pmid: 10508632
references.citation: Protein Expr Purif. 1997 Oct;11(1):47-52
references.pmid: 9325138
references.citation: Methods Enzymol. 2009;463:131-47
references.pmid: 19892171
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1108-18
references.pmid: 21964262
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):411-21
references.pmid: 10508629
references.citation: Protein Expr Purif. 1996 Mar;7(2):129-36
references.pmid: 8812844
references.citation: Biochemistry. 1991 Jul 30;30(30):7527-34
references.pmid: 1854752
references.citation: Cell Mol Life Sci. 2004 Jun;61(12):1455-74
references.pmid: 15197470
references.citation: Protein Expr Purif. 1998 Mar;12(2):159-65
references.pmid: 9518456
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1140-57
references.pmid: 21983546
references.citation: Bioprocess Technol. 1991;12:57-83
references.pmid: 1367090
references.citation: Structure. 2014 Sep 2;22(9):1311-1321
references.pmid: 25132083
references.citation: Curr Opin Struct Biol. 2000 Feb;10(1):40-5
references.pmid: 10679456
references.citation: Protein Sci. 1999 Jul;8(7):1475-83
references.pmid: 10422836
references.citation: Biotechnology (N Y). 1991 Aug;9(8):725-30
references.pmid: 1367632
references.citation: Bioessays. 1985 Jul;3(1):27-31
references.pmid: 3024631
references.citation: Anal Biochem. 1986 Jun;155(2):215-42
references.pmid: 3524314
references.citation: Curr Opin Struct Biol. 2002 Feb;12(1):54-60
references.pmid: 11839490
references.citation: Protein Expr Purif. 2001 Mar;21(2):260-7
references.pmid: 11237687
references.citation: Protein Eng. 2000 Jan;13(1):67-71
references.pmid: 10679532
references.citation: Biochem J. 1999 Mar 1;338 ( Pt 2):335-42
references.pmid: 10024508
references.citation: Biotechnology (N Y). 1991 Feb;9(2):157-62
references.pmid: 1369317
references.citation: Microb Cell Fact. 2012 Jun 11;11:76
references.pmid: 22686540
references.citation: J Biol Chem. 1982 Sep 25;257(18):10766-9
references.pmid: 6955305
references.citation: J Biotechnol. 2000 Jan 21;76(2-3):97-119
references.pmid: 10656326
references.citation: Protein Eng. 1987 Feb-Mar;1(2):141-6
references.pmid: 3507696
references.citation: Protein Expr Purif. 2002 Apr;24(3):412-9
references.pmid: 11922757
references.citation: Curr Opin Biotechnol. 2001 Apr;12(2):202-7
references.pmid: 11287238
references.citation: J Mol Biol. 1991 Dec 20;222(4):865-8
references.pmid: 1762154
references.citation: J Bacteriol. 1997 Apr;179(8):2465-71
references.pmid: 9098040
references.citation: Curr Opin Biotechnol. 1996 Aug;7(4):409-16
references.pmid: 8768899
references.citation: Biochemistry. 2000 Feb 22;39(7):1870-8
references.pmid: 10677238
references.citation: J Mol Biol. 1992 Apr 20;224(4):1075-85
references.pmid: 1569568
references.citation: Structure. 1996 Mar 15;4(3):239-42
references.pmid: 8805540
references.citation: Nat Methods. 2008 Feb;5(2):135-46
references.pmid: 18235434
references.citation: Biochem J. 1992 Aug 1;285 ( Pt 3):871-9
references.pmid: 1497625
references.citation: J Biol Chem. 1989 Jul 15;264(20):11539-42
references.pmid: 2473064
references.citation: Biochem Biophys Res Commun. 1978 Mar 30;81(2):416-21
references.pmid: 208522
references.citation: Curr Opin Biotechnol. 1999 Aug;10(4):376-81
references.pmid: 10449317
references.citation: Curr Opin Biotechnol. 1998 Oct;9(5):497-501
references.pmid: 9821278
references.citation: Curr Opin Struct Biol. 2001 Feb;11(1):114-9
references.pmid: 11179900
references.citation: Nat Methods. 2006 Jan;3(1):55-64
references.pmid: 16369554
references.citation: Biochemistry. 1975 Oct 21;14(21):4634-8
references.pmid: 1101952
references.citation: Curr Opin Struct Biol. 1999 Feb;9(1):102-10
references.pmid: 10047582
references.citation: Nat Rev Mol Cell Biol. 2013 Oct;14(10):630-42
references.pmid: 24026055
references.citation: J Virol. 2002 Mar;76(6):2692-702
references.pmid: 11861836
references.citation: Methods Mol Biol. 2012;831:71-84
references.pmid: 22167669
references.citation: Biotechnol Adv. 2012 Sep-Oct;30(5):1102-7
references.pmid: 21968145
references.citation: Science. 1991 May 3;252(5006):698-702
references.pmid: 1902591
references.citation: Protein Eng. 1987 Oct-Nov;1(5):413-7
references.pmid: 3509874
references.citation: Protein Sci. 2002 Feb;11(2):313-21
references.pmid: 11790841
references.citation: Biochemistry. 1995 Apr 18;34(15):4919-32
references.pmid: 7711014
references.citation: Biotechnology (N Y). 1993 Feb;11(2):187-93
references.pmid: 7763371
references.citation: Curr Opin Biotechnol. 2001 Apr;12(2):195-201
references.pmid: 11287237
references.citation: Protein Sci. 1993 Nov;2(11):1862-8
references.pmid: 8268797
references.citation: Protein Expr Purif. 2011 Jul;78(1):1-5
references.pmid: 20851184
references.citation: Protein Expr Purif. 1993 Oct;4(5):425-33
references.pmid: 8251755
references.citation: Cell. 1999 Nov 12;99(4):433-42
references.pmid: 10571185
references.citation: Biotechnology (N Y). 1991 Mar;9(3):273-8
references.pmid: 1367302
references.citation: Nat Biotechnol. 2004 Jul;22(7):877-82
references.pmid: 15195104
references.citation: J Biol Chem. 1978 May 25;253(10):3453-8
references.pmid: 649582
references.citation: Curr Opin Biotechnol. 2000 Oct;11(5):450-4
references.pmid: 11024362
references.citation: Protein Sci. 1996 Mar;5(3):495-506
references.pmid: 8868486
references.citation: Nature. 1993 May 13;363(6425):172-6
references.pmid: 8483502
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):428-33
references.pmid: 10508635
references.citation: FEBS Lett. 1991 May 20;283(1):135-9
references.pmid: 2037066
references.citation: Science. 1992 Jan 17;255(5042):306-12
references.pmid: 1549776
references.citation: Biochemistry. 1996 Aug 13;35(32):10256-61
references.pmid: 8756680
references.citation: Protein Sci. 1997 Aug;6(8):1653-60
references.pmid: 9260278
references.citation: Methods Enzymol. 1990;182:264-76
references.pmid: 2179665
references.citation: Nat Struct Biol. 1996 Apr;3(4):340-5
references.pmid: 8599760
references.citation: Methods Enzymol. 1986;119:166-77
references.pmid: 3531764
references.citation: Proc Natl Acad Sci U S A. 1987 May;84(9):2718-22
references.pmid: 3106976
references.citation: Ann N Y Acad Sci. 1994 May 2;721:348-64
references.pmid: 8010684
references.citation: Curr Opin Biotechnol. 1999 Apr;10(2):156-9
references.pmid: 10209142
references.citation: Methods Enzymol. 1999;309:48-58
references.pmid: 10507015
references.citation: Trends Biotechnol. 2005 Jun;23(6):316-20
references.pmid: 15922084
references.citation: J Biol Chem. 2000 Jun 23;275(25):18619-22
references.pmid: 10781576
references.citation: EMBO J. 1990 May;9(5):1665-72
references.pmid: 2184035
references.citation: Science. 1999 Aug 13;285(5430):1021-2
references.pmid: 10475844
references.citation: J Biol Chem. 1979 May 10;254(9):3206-10
references.pmid: 372187
references.citation: Biochemistry. 1994 Aug 16;33(32):9405-13
references.pmid: 8068616
references.citation: Gene. 1988 May 30;65(2):259-68
references.pmid: 2970420
references.citation: Experientia. 1992 Feb 15;48(2):178-201
references.pmid: 1740190
references.citation: Biotechniques. 2001 Dec;31(6):1254, 1256, 1258
references.pmid: 11768651
references.citation: Science. 1967 Jun 16;156(3781):1451-5
references.pmid: 4304946
references.citation: Anal Biochem. 1994 Nov 15;223(1):1-6
references.pmid: 7695083
references.citation: Protein Expr Purif. 1997 Oct;11(1):1-16
references.pmid: 9325133
references.citation: Curr Opin Struct Biol. 2000 Oct;10(5):585-92
references.pmid: 11042458
references.citation: Adv Protein Chem. 1968;23:121-282
references.pmid: 4882248
references.citation: Protein Sci. 1995 Nov;4(11):2411-23
references.pmid: 8563639
references.citation: Protein Expr Purif. 2000 Dec;20(3):435-43
references.pmid: 11087683
references.citation: Curr Opin Biotechnol. 1992 Aug;3(4):363-9
references.pmid: 1368437
references.citation: Biochemistry. 1984 Dec 18;23(26):6474-80
references.pmid: 6099140
references.citation: J Chromatogr. 1987 Jan 30;387:291-300
references.pmid: 3494032