Robustness of hen lysozyme monitored by random mutations.
We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg(2+) and Mn(2+) on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.
Kunichika
Kaori
K
Graduate School of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan.
Hashimoto
Yoshio
Y
Imoto
Taiji
T
D000596:Amino Acids / Q000235:genetics; D000818:Animals; D020137:Base Pair Mismatch; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015723:Gene Library; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019914:Taq Polymerase / Q000235:genetics
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D008345:Manganese; D019914:Taq Polymerase; D009113:Muramidase; D008274:Magnesium