Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling

被引:106
作者
Christians, FC
Scapozza, L
Crameri, A
Folkers, G
Stemmer, WPC
机构
[1] Maxygen Inc, Santa Clara, CA 95051 USA
[2] Swiss Fed Inst Technol, Dept Pharm, ETH Zurich, CH-8057 Zurich, Switzerland
关键词
random mutagenesis; sexual PCR/DNA shuffling; suicide gene; gene transfer; zidovudine;
D O I
10.1038/7003
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high throughput robotic screen identified chimeras with an enhanced ability to phosphoryl;ate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced Kw for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.
引用
收藏
页码:259 / 264
页数:6
相关论文
共 33 条
[1]   Improving AZT efficacy [J].
Balzarini, J ;
Degrève, B ;
De Clercq, E .
NATURE MEDICINE, 1998, 4 (02) :132-132
[2]   Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy [J].
Black, ME ;
Newcomb, TG ;
Wilson, HMP ;
Loeb, LA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (08) :3525-3529
[3]   IDENTIFICATION OF IMPORTANT RESIDUES WITHIN THE PUTATIVE NUCLEOSIDE BINDING-SITE OF HSV-1 THYMIDINE KINASE BY RANDOM SEQUENCE SELECTION - ANALYSIS OF SELECTED MUTANTS IN-VITRO [J].
BLACK, ME ;
LOEB, LA .
BIOCHEMISTRY, 1993, 32 (43) :11618-11626
[4]  
Bouayadi K, 1997, CANCER RES, V57, P110
[5]   CRYSTAL-STRUCTURES OF THE THYMIDINE KINASE FROM HERPES-SIMPLEX VIRUS TYPE-I IN COMPLEX WITH DEOXYTHYMIDINE AND GANCICLOVIR [J].
BROWN, DG ;
VISSE, R ;
SANDHU, G ;
DAVIES, A ;
RIZKALLAH, PJ ;
MELITZ, C ;
SUMMERS, WC ;
SANDERSON, MR .
NATURE STRUCTURAL BIOLOGY, 1995, 2 (10) :876-881
[6]   Improved properties of FLP recombinase evolved by cycling mutagenesis [J].
Buchholz, F ;
Angrand, PO ;
Stewart, AF .
NATURE BIOTECHNOLOGY, 1998, 16 (07) :657-662
[7]  
CHAMPNESS JN, 1998, STRUCT FUNCT GENET, V32, P350
[8]  
Cleland W W, 1979, Methods Enzymol, V63, P103
[9]   Improved green fluorescent protein by molecular evolution using DNA shuffling [J].
Crameri, A ;
Whitehorn, EA ;
Tate, E ;
Stemmer, WPC .
NATURE BIOTECHNOLOGY, 1996, 14 (03) :315-319
[10]   DNA shuffling of a family of genes from diverse species accelerates directed evolution [J].
Crameri, A ;
Raillard, SA ;
Bermudez, E ;
Stemmer, WPC .
NATURE, 1998, 391 (6664) :288-291