Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.
Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.
Seelig
Burckhard
B
Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology (CCIB), 7215 Simches Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA.
Szostak
Jack W
JW
D000595:Amino Acid Sequence; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D011130:RNA Ligase (ATP) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D016335:Zinc Fingers
D004798:Enzymes; D008024:Ligands; D019151:Peptide Library; D012333:RNA, Messenger; D011130:RNA Ligase (ATP)
Nat Biotechnol. 2003 Jun;21(6):679-85
12754520
PCR Methods Appl. 1992 Aug;2(1):28-33
1490172
Mol Divers. 1997;2(3):156-64
9238646
Bioorg Med Chem. 2004 Oct 15;12(20):5247-68
15388154
Curr Opin Biotechnol. 2000 Aug;11(4):338-53
10975453
Science. 2004 Jun 25;304(5679):1967-71
15218149
J Biol Chem. 1994 Oct 7;269(40):25120-8
7929200
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12297-302
9356443
Science. 2006 Jan 27;311(5760):535-8
16439663
Anal Biochem. 1977 Nov;83(1):228-39
920942
Nature. 2001 Apr 5;410(6829):715-8
11287961
Science. 1993 Sep 10;261(5127):1411-8
7690155
Chem Biol. 2003 Jan;10(1):69-79
12573700
Chem Biol. 2002 Feb;9(2):253-64
11880040
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3750-5
11274392
J Am Chem Soc. 2005 Sep 28;127(38):13124-5
16173722
Chem Biol. 2006 Feb;13(2):139-47
16492562
J Mol Biol. 2000 Mar 24;297(2):309-19
10715203
Curr Opin Biotechnol. 2004 Aug;15(4):272-9
15357999
Curr Opin Struct Biol. 2005 Aug;15(4):447-52
16006119
Science. 1992 May 15;256(5059):992-7
1589782
Nucleic Acids Res. 2000 Sep 15;28(18):E83
10982894
Science. 1995 Jul 21;269(5222):364-70
7618102
Curr Opin Biotechnol. 2003 Aug;14(4):438-43
12943855
Chem Biol. 2002 Aug;9(8):933-42
12204693
J Mol Biol. 1998 Aug 14;281(2):271-84
9698548
J Mol Recognit. 2002 May-Jun;15(3):126-34
12203838
Curr Opin Chem Biol. 2005 Apr;9(2):210-6
15811807
J Am Chem Soc. 1996 Apr 10;118(14):3332-9
11539267
Methods Enzymol. 2000;318:268-93
10889994
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14274-9
11724958
Biochemistry. 1997 Aug 26;36(34):10498-505
9265630
Nature. 2001 Jan 11;409(6817):258-68
11196655
Nucleic Acids Res. 1988 Nov 25;16(22):10881-90
2849754