title: Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges.
abstract: We pursued several strategies for expressing either full-length Sus scrofa diacylglycerol kinase (DGK) alpha or the catalytic domain (alphacat) in Escherichia coli. Alphacat could be extracted, refolded, and purified from inclusion bodies, but when subjected to analytical gel filtration chromatography, it elutes in the void volume, in what we conclude are microscopic aggregates unsuitable for x-ray crystallography. Adding glutathione S-transferase, thioredoxin, or maltose binding protein as N-terminal fusion tags did not improve alphacat's solubility. Coexpressing with bacterial chaperones increased the yield of alphacat in the supernatant after high-speed centrifugation, but the protein still elutes in the void upon analytical gel filtration chromatography. We believe our work will be of interest to those interested in the structure of eukaryotic DGKs, so that they know which expression strategies have already been tried, as well as to those interested in protein folding and those interested in chaperone/target-protein interactions.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Petro
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Raben
mesh_terms: D000818:Animals; D002384:Catalysis; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D034421:Sus scrofa / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019852:Diacylglycerol Kinase
references.citation: Microb Cell Fact. 2010 Sep 02;9:64
references.pmid: 20813055
references.citation: Neurosci Lett. 2004 Dec 6;372(3):190-5
references.pmid: 15542238
references.citation: Lipids. 1980 May;15(5):285-91
references.pmid: 6248716
references.citation: Biochem Biophys Res Commun. 2012 Jul 6;423(3):571-6
references.pmid: 22695121
references.citation: J Neurochem. 1993 Apr;60(4):1212-9
references.pmid: 8384245
references.citation: Nature. 1990 Mar 22;344(6264):345-8
references.pmid: 2156169
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt 3):295-7
references.pmid: 16511327
references.citation: Arch Biochem Biophys. 1981 Jun;209(1):266-75
references.pmid: 6269496
references.citation: Oncogene. 2007 Aug 2;26(35):5154-62
references.pmid: 17334400
references.citation: Biochem J. 2002 Dec 15;368(Pt 3):809-15
references.pmid: 12296770
references.citation: Bioinformatics. 2007 Oct 1;23(19):2536-42
references.pmid: 17150993
references.citation: Biochem Cell Biol. 1989 Apr-May;67(4-5):233-41
references.pmid: 2550036
references.citation: J Biol Chem. 1990 Nov 5;265(31):19237-43
references.pmid: 2172248
references.citation: Biochemistry. 1999 Mar 16;38(11):3310-9
references.pmid: 10079074
references.citation: Methods Enzymol. 2000;326:254-70
references.pmid: 11036647
references.citation: Proc Natl Acad Sci U S A. 1986 Dec;83(24):9378-82
references.pmid: 3025839
references.citation: J Biol Chem. 1997 Jun 20;272(25):15607-12
references.pmid: 9188448
references.citation: Appl Biochem Biotechnol. 1997 Jun;66(3):197-238
references.pmid: 9276922
references.citation: Biochemistry. 2005 Aug 2;44(30):10199-207
references.pmid: 16042397
references.citation: J Biol Chem. 1999 Jul 23;274(30):21251-6
references.pmid: 10409682
references.citation: Lipids. 1986 Mar;21(3):206-11
references.pmid: 3010026
references.citation: J Biol Chem. 2001 Feb 23;276(8):5900-7
references.pmid: 11078732
references.citation: J Biol Chem. 1991 Apr 15;266(11):7096-100
references.pmid: 1849900
references.citation: J Biol Chem. 2012 Dec 7;287(50):41619-27
references.pmid: 23091060
references.citation: J Biol Chem. 1995 Dec 1;270(48):28647-53
references.pmid: 7499383
references.citation: Nature. 1999 Nov 11;402(6758):147-54
references.pmid: 10647006
references.citation: Microbiol Mol Biol Rev. 2002 Mar;66(1):64-93; table of contents
references.pmid: 11875128
references.citation: Biochem Biophys Res Commun. 1991 Dec 31;181(3):1015-21
references.pmid: 1662491
references.citation: Pharmacol Ther. 2007 Sep;115(3):352-9
references.pmid: 17659347
references.citation: Nat Protoc. 2007;2(10):2632-9
references.pmid: 17948006
references.citation: J Cell Biol. 2001 Mar 19;152(6):1135-43
references.pmid: 11257115
references.citation: Curr Opin Biotechnol. 2006 Aug;17(4):353-8
references.pmid: 16781139
references.citation: Biochim Biophys Acta. 1992 Jul 31;1122(2):219-24
references.pmid: 1322704
references.citation: Comp Funct Genomics. 2004;5(4):342-53
references.pmid: 18629169
references.citation: J Biol Chem. 2005 Oct 14;280(41):34888-99
references.pmid: 16081412
references.citation: FEBS Lett. 1990 Nov 26;275(1-2):151-8
references.pmid: 2175712
references.citation: Curr Protoc Protein Sci. 2004 Nov;Chapter 6:6.3.1-6.3.18
references.pmid: 18429271
references.citation: J Biol Chem. 1992 Jan 15;267(2):842-7
references.pmid: 1309777
references.citation: Mol Biol Cell. 2011 Mar 15;22(6):817-30
references.pmid: 21248205
references.citation: Biochem J. 1996 Sep 1;318 ( Pt 2):583-90
references.pmid: 8809050
references.citation: Protein Expr Purif. 2009 Apr;64(2):185-93
references.pmid: 19038347
references.citation: Structure. 2008 Mar;16(3):380-7
references.pmid: 18334213
references.citation: Biochem Pharmacol. 1995 Jul 17;50(2):235-41
references.pmid: 7632168
references.citation: J Biol Chem. 1990 Jan 15;265(2):794-800
references.pmid: 2153114
references.citation: Protein Expr Purif. 2011 Apr;76(2):197-204
references.pmid: 21111047
references.citation: J Cell Biol. 2001 Apr 2;153(1):207-20
references.pmid: 11285286
references.citation: FEBS Lett. 1989 Sep 25;255(2):409-13
references.pmid: 2551742
references.citation: J Biol Chem. 1984 Sep 25;259(18):11197-202
references.pmid: 6088535
references.citation: Chem Phys Lipids. 2013 Jan;166:26-30
references.pmid: 23261795
references.citation: J Cell Biol. 2003 Mar 17;160(6):929-37
references.pmid: 12629049
references.citation: Biochem J. 1993 Feb 1;289 ( Pt 3):875-81
references.pmid: 7679574
references.citation: Biochem Biophys Res Commun. 2005 Aug 5;333(3):686-93
references.pmid: 15961060
references.citation: Biochemistry. 2007 May 22;46(20):6109-17
references.pmid: 17455907
references.citation: J Biol Chem. 2004 Feb 27;279(9):8230-41
references.pmid: 14665624
references.citation: Structure. 2008 Jul;16(7):1036-46
references.pmid: 18611377
references.citation: Gene. 1988 Jul 15;67(1):31-40
references.pmid: 3047011
references.citation: Nature. 1992 Jul 9;358(6382):169-70
references.pmid: 1614549
references.citation: J Antibiot (Tokyo). 1994 Apr;47(4):499-501
references.pmid: 8195052
references.citation: Bioinformatics. 2009 Sep 1;25(17):2200-7
references.pmid: 19549632
references.citation: BMC Biotechnol. 2004 Dec 14;4:32
references.pmid: 15598350
references.citation: Biochem J. 1997 Jan 1;321 ( Pt 1):59-64
references.pmid: 9003401
references.citation: Proteins. 2007 Jul 1;68(1):13-25
references.pmid: 17393457
references.citation: FEBS Lett. 1986 May 26;201(1):97-100
references.pmid: 3011508
references.citation: J Biol Chem. 2009 Nov 6;284(45):31062-73
references.pmid: 19744926
references.citation: Arch Biochem Biophys. 2001 Aug 15;392(2):219-25
references.pmid: 11488595
references.citation: RNA. 2004 Nov;10(11):1751-63
references.pmid: 15496522
references.citation: Biochem Pharmacol. 2000 Apr 1;59(7):763-72
references.pmid: 10718334
references.citation: Biochemistry. 2012 Sep 18;51(37):7250-62
references.pmid: 22924672
references.citation: Biotechnology (N Y). 1993 Feb;11(2):187-93
references.pmid: 7763371
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Biochem J. 1990 Feb 15;266(1):291-9
references.pmid: 2155609
references.citation: Protein Expr Purif. 2011 Sep;79(1):16-24
references.pmid: 21640828
references.citation: J Biol Chem. 1983 Feb 10;258(3):1767-74
references.pmid: 6296111
references.citation: Methods Enzymol. 2000;326:312-21
references.pmid: 11036650
references.citation: Curr Opin Biotechnol. 1995 Oct;6(5):494-500
references.pmid: 7579660
references.citation: Biochem J. 1991 Nov 15;280 ( Pt 1):233-41
references.pmid: 1660266
references.citation: J Mol Biol. 1996 Aug 23;261(3):328-33
references.pmid: 8780775
references.citation: Plant Mol Biol. 1996 Feb;30(3):647-53
references.pmid: 8605313
references.citation: Biochemistry. 1999 Mar 16;38(11):3320-6
references.pmid: 10079075
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D348-52
references.pmid: 23197659
references.citation: BMB Rep. 2011 Apr;44(4):279-84
references.pmid: 21524355
references.citation: Biochem Soc Trans. 1993 Nov;21(4):490S
references.pmid: 8132057
references.citation: J Biol Chem. 1994 Aug 19;269(33):21155-64
references.pmid: 8063736
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: J Biol Chem. 2005 Dec 2;280(48):39870-81
references.pmid: 16210324
references.citation: Biochem J. 1990 Dec 15;272(3):569-75
references.pmid: 2176468
references.citation: Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2874-8
references.pmid: 1826368
references.citation: Plant Physiol. 1992 Mar;98(3):1148-53
references.pmid: 16668739
references.citation: J Biol Chem. 2000 Nov 3;275(44):34092-9
references.pmid: 10918059
references.citation: Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6137-41
references.pmid: 1648730
references.citation: BMC Biotechnol. 2007 Jun 12;7:32
references.pmid: 17565681
references.citation: Biochem J. 1989 Mar 1;258(2):455-62
references.pmid: 2539807
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3119-24
references.pmid: 17360617