title: Highly diverse protein library based on the ubiquitous (β/α)₈ enzyme fold yields well-structured proteins through in vitro folding selection.
abstract: Proper protein folding is a prerequisite for protein stability and enzymatic activity. Although directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (β/α)₈ barrel fold, which is found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)₈ barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants, and then combined them to assemble the final library (10¹⁴ DNA sequences). The resulting library was analyzed by using the in vitro protease assay and an in vivo GFP-folding assay; it contains ∼10¹² soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)₈-barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput mRNA display method with selection for folding. The resulting (β/α)₈ barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)₈ fold, and to isolate novel enzymes.
authors: BioTechnology Institute & Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Twin-Cities, 1479 Gortner Ave, St. Paul, MN 55108 (USA).
authors: Misha V
authors: MV
authors: Golynskiy
authors: John C
authors: JC
authors: Haugner
authors: Burckhard
authors: B
authors: Seelig
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation; D008958:Models, Molecular; D019151:Peptide Library; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D019151:Peptide Library; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase
keywords: (beta/alpha)8 barrel; GFP-folding reporter; mRNA display; protein engineering; protein folding
references: Protein Sci. 2010 Oct;19(10):1817-9
references: 20717908
references: J Mol Biol. 2002 Aug 30;321(5):741-65
references: 12206759
references: J Mol Biol. 2004 Apr 2;337(4):871-9
references: 15033357
references: Curr Opin Struct Biol. 2009 Oct;19(5):596-604
references: 19765975
references: Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5869-74
references: 16581913
references: Science. 2006 Jan 27;311(5760):535-8
references: 16439663
references: Methods Mol Biol. 2010;634:217-32
references: 20676987
references: Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12297-302
references: 9356443
references: Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16152-7
references: 17913881
references: FEBS Lett. 2001 Mar 16;492(3):193-8
references: 11257493
references: Nature. 2007 Aug 16;448(7155):828-31
references: 17700701
references: Curr Opin Struct Biol. 2005 Aug;15(4):472-8
references: 16043338
references: Proc Natl Acad Sci U S A. 2012 Mar 6;109(10):3790-5
references: 22357762
references: Protein Sci. 2004 Mar;13(3):752-62
references: 14767075
references: Science. 2010 Jul 16;329(5989):309-13
references: 20647463
references: Chem Rev. 2005 Nov;105(11):4038-55
references: 16277370
references: J Mol Biol. 2011 Aug 5;411(1):143-57
references: 21635898
references: Nat Protoc. 2011 Apr;6(4):540-52
references: 21455189
references: Methods Mol Biol. 2013;978:73-92
references: 23423890
references: Biochemistry. 2005 Sep 6;44(35):11722-9
references: 16128573
references: Protein Eng Des Sel. 2012 Jun;25(6):285-93
references: 22490958
references: Nat Biotechnol. 1998 Oct;16(10):955-60
references: 9788353
references: Chembiochem. 2011 Jul 4;12(10):1501-7
references: 21472838
references: J Mol Biol. 1965 Sep;13(2):482-95
references: 5867031
references: Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11161-6
references: 10500147
references: J Biol Chem. 2012 Jan 2;287(1):3-10
references: 22069330
references: J Mol Biol. 2012 Feb 10;416(1):21-32
references: 22197379
references: Annu Rev Biochem. 2010;79:471-505
references: 20235827
references: Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16448-53
references: 15539462
references: Nature. 2012 May 09;485(7397):185-94
references: 22575958
references: EMBO J. 2003 Jan 2;22(1):24-35
references: 12505981
references: Nat Chem Biol. 2013 Feb;9(2):81-3
references: 23222886
references: FEBS Lett. 2003 Mar 27;539(1-3):24-8
references: 12650920
references: Chem Biol. 2006 Feb;13(2):139-47
references: 16492562
references: J Mol Biol. 2000 Mar 24;297(2):309-19
references: 10715203
references: Biochemistry. 2012 Aug 14;51(32):6463-75
references: 22809162
references: Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3704-9
references: 19237570
references: J Mol Biol. 2009 Apr 10;387(4):949-64
references: 19233201
references: Orig Life Evol Biosph. 2004 Feb;34(1-2):151-7
references: 14979652
references: Science. 2006 Jan 27;311(5760):475-6
references: 16439649
references: Protein Eng Des Sel. 2013 Mar;26(3):181-6
references: 23175796
references: Nat Biotechnol. 2002 Sep;20(9):927-32
references: 12205510
references: Curr Opin Chem Biol. 2003 Apr;7(2):252-64
references: 12714059
references: J Mol Biol. 2012 Feb 17;416(2):255-70
references: 22226942
references: Biochemistry. 1988 Feb 23;27(4):1158-67
references: 3365378
references: Science. 2008 Mar 7;319(5868):1387-91
references: 18323453
references: Proteins. 2004 Jul 1;56(1):167-70
references: 15162496
references: J Am Chem Soc. 2008 Jan 9;130(1):176-85
references: 18067292
references: Trends Biotechnol. 2010 Jul;28(7):340-5
references: 20483496
references: J Mol Biol. 2010 Nov 5;403(4):562-77
references: 20850457
references: Nature. 2008 May 8;453(7192):190-5
references: 18354394
references: Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):606-11
references: 15644440