Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.
Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady-state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.
Chapman
D
D
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow 83844, USA.
Hochstrasser
R
R
Millar
D
D
Youderian
P
P
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis; D001426:Bacterial Proteins; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D007930:Leucine; D008715:Methionine; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan; D029968:Escherichia coli Proteins
D001059:Apoproteins; D001426:Bacterial Proteins; D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D007930:Leucine; D008715:Methionine; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins