COMBINATORIAL MUTAGENESIS OF 3 MAJOR GROOVE-CONTACTING RESIDUES OF ECORI - SINGLE AND DOUBLE AMINO-ACID REPLACEMENTS RETAINING METHYLTRANSFERASE-SENSITIVE ACTIVITIES

被引：25

作者：

OSUNA, J ^{[1
]}

FLORES, H ^{[1
]}

SOBERON, X ^{[1
]}

机构：

[1] UNIV NACL AUTONOMA MEXICO,CTR INVEST INGN GENET & BIOTECNOL,APDO POSTAL 510-3,CUERNAVACA 62271,MORELOS,MEXICO

来源：

GENE | 1991年 / 106卷 / 01期

关键词：

DNA-PROTEIN INTERACTIONS; ENZYME SPECIFICITY; POLYMERASE CHAIN REACTION; PROTEIN ENGINEERING;

D O I：

10.1016/0378-1119(91)90559-T

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

A library of mutant ecoRIR genes encoding EcoRI restriction endonuclease was generated using trinucleotide blocks and a combination of recombinant DNA procedures, including primer extension and the polymerase chain reaction. Codons corresponding to three amino acids (E144, R145 and R200), previously implicated in the specific recognition of the DNA substrate, were combinatorially mutated so as to generate a library that potentially contains all 20(3) possible single, double and triple aa replacements, in a balanced distribution. Inspection of the phenotypes of Escherichia coli colonies bearing the mutant genes showed that several of them retained activities that were deleterious to the cells but were still protected by the EcoRI methyltransferase. These included new enzyme variants, including non-conservative single (Thr or Val for Glu144) and double (Val for Glu144 and Thr for Arg145 ) replacements.

引用

页码：7 / 12

页数：6

共 21 条

[11] THE ENERGETIC BASIS OF SPECIFICITY IN THE ECO RI ENDONUCLEASE DNA INTERACTION [J].