Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis

FEBS Letters

Patthy, András²  Sprengel, Gunther³  Gráf, László¹  Pál, Gábor¹ 

[1] Department of Biochemistry, Eötvös University, H-1088 Puskin u. 3., Budapest, Hungary;Agricultural Biotechnology Center, H-2100 Gödöllö, P.O.B. 170., Hungary;Max-Planck-Institut für Biochemie, D-8033 Martinsried, Germany

关键词: Ecotin;    Serine proteinase inhibitor;    Site-directed mutagenesis;    PCR;    polymerase chain reaction;    dut;    dUTPase;    ung;    uracil N-glicosylase;    TPCK;    N-tosyl-l-phenylalanine chloromethyl ketone;    TLCK;    N-p-tosyl-l-lysine chloromethyl ketone;    IPTG;    Isopropyl-β-d-thiogalactopyranoside;    MUB;    4-methylumbelliferone;    MUGB;    4-methylumbelliferyl-p-guanidinobenzoate;    MUTMAC;    4-methylum-belliferyl-p-trimethylammonium cinnamate chloride;    AMC;    7-amino-4-methylcoumarin;   

DOI  :  10.1016/0014-5793(94)80584-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The gene of ecotin, an E. coli proteinase inhibitor, was cloned, and by site-directed mutagenesis the active site residue of the protein, Met⁸⁴, was mutated to Lys, Arg and Leu. The recombinant wild-type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.

【 授权许可】

Unknown