TABLE 1.

Mutagenic primers used for site-directed mutagenesis

NameaNucleotide sequencebCodon changeRestriction site
R8A5′-CCCTCTGGTCTGCAGTCGTGAG-3′CGA→GCA PstI
R8K5′-CACCAACCCTCTGGTCCAAGGTCGTGAGATTTGG-3′CGA→AAG EcoT14I
R8Q5′-CACCAACCCTCTGGTCGCAGGTCGTGAGATTTGG-3′CGA→CAG BspMIc
R8L5′-CACCAACCCTCTGGTCCTTGGTCGTGAGATTTGG-3′GCA→TTG EcoT14I
R11A5′-GAGTCGTGGCATTCGGCTCTGG-3′AGA→GCA Mva1269I
H30A5′-TCACCACCACTGCAGTTATACCAA-3′CAT→GCA PstI
H30Y5′-TTTATCACCACTACGTATGTTATACCA-3′CAT→TAT BsaAI
H30Q5′-TCACCACCACGCAGGTTATACCAA-3′CAT→CAG BspMIc
H30N5′-TTTATCACCACGACCAATGTCATACCAACTGG-3′CAT→AAT PshAIc
H30E5′-TTTATCACCACGACCGAAGTCATACCAACTGG-3′CAT→GAA PshAIc
H30D5′-CACCACCACCGACGTCATACCAACTG-3′CAT→GAC AatII
H30R5′-TCACCACCACGCGTGTTATACCA-3′CAT→CGT MluI
R37A5′-CTGGGGTGGCTGAGTTCTTTGG-3′CGT→GCT(EcoRI)d
E38A5′-GGGTGCGTGCATTCTTTGG-3′GAA→GCA(EcoRI)d
E42A5′-TCTTTGGGGCGCCCATTGA-3′GAG→GCG EheI
E45A5′-GGAGCCCATTGCTAGCATAGCAATCCA-3′GAA→GCT NheI
H50A5′-TAGCAATCGCTCGAGCTGGTGA-3′CAT→GCT XhoI
R51A5′-CAATCCATGCTGCAGGTGAATT-3′CGT→GCT PstI
E54A5′-CGTGCTGGCGCCTTTACACA-3′GAA→GCC EheI
R59A5′-CACAATTCGCATTCTCACGCAA-3′AGG→GCA Mva1269I
R62A5′-GGTTTTCAGCCAAGGTCCGCCC-3′CGC→GCC EcoT14I
K63A5′-AGGTTTTCCCGGGCAGTCCGCC-3′AAA→GCA SmaI
R65A5′-ACGCAAAGTGGCGCCGGATCTG-3′CGC→GCG EheI
D67A5′-AAAGTCCGGCCGGCTCTGACTG-3′GAT→GCT NaeI
E74A5′-GAATGGTGCTAGCGGAAGGCT-3′GAG→GCG NheI
E75A5′-TGTTGGAGGCCGGCTGTCCT-3′GAA→GCC NaeI
E79A5′-GCTGTCCTGCAGGTGTCGTG-3′GAG→GCA PstI
K88A5′-TTCTTATCGCTCGAGACTCTGG-3′AAG→GCT XhoI
K88R5′-CTATTCTTATCCGTCGCGACTCTGGTGA-3′AAG→CGT NruI
K88Q5′-TATTCTTATCCAGCGGGACTCTGGTG-3′AAG→CAG MspA1I
K88L5′-CTATTCTTATCCTTCGCGACTCTGGTGA-3′AAG→CTT NruI
R89A5′-TTATCAAGGCAGACTCTGGT-3′CGT→GCA AlwNI
R89K5′-TTCTTATCAAGAAAGACTCGGGTGAGCTGCT-3′CGT→AAA AvaI
R89Q5′-TCTTATCAAGCAGGACTCGGGTGAGCTGCT-3′CGT→CAG AvaI
R89L5′-CTTATCAAGCTTGACTCTGGT-3′CGT→CTT HindIII
D90A5′-TATCAAGCGTGCTAGCGGTGAGCTGCT-3′GAC→GCT NheI
E93A5′-TGACTCTGGTGCACTGCTACCCCT-3′GAG→GCA ApaLI
R100A5′-CCCTTGCTGTCGCGATGGGCGCTAT-3′CGA→GCG NruI
K108A5′-GCGTCCATGGCGATCCAGGG-3′AAG→GCG NcoI
R112A5′-GATCCAGGGCGCCCTAGTGCAT-3′AGG→GCC EheI
H115A5′-GGCTAGTGGCCGGCCAATCTGG-3′CAT→GCC NaeI
K128A5′-GCTAACGCTGCAGGGATGGAT-3′AAG→GCA PstI
D131A5′-GGGGATGGCCTTGGGTACCCT-3′GAT→GCC EcoT14I
D138A5′-CTACCAGGCGCCTGTGGTGC-3′GAT→GCC EheI
D138E5′-TACCAGGTGAATGCGGTGCCCCTT-3′GAT→GAA Mva1269I
D138N5′-CCCTACCAGGCAATTGTGGTGC-3′GAT→AAT MunI
D138M5′-AGGTACCCTACCCGGGATGTGTGTGGTGCCCCT-3′GAT→ATG SmaI
C139Ae 5′-CTACCAGGTGATGCCGGCGCCCCTTATGTG-3′TGT→GCC EheI
C139S5′-CCAGGTGATTCCGGAGCCCCTTATG-3′TGT→TCC AccIII
C139T5′-TACCAGGTGATACCGGTGCCCCTTA-3′TGT→ACC BshTI
C139Y5′-CCAGGTGATTATGGCGCCCCTTATG-3′TGT→TAT EheI
C139M5′-TACCAGGTGATATGGGGCGCCCCTTATGT-3′TGT→ATG EheI
K146A5′-ATGTGTACGCACGTAACAATGA-3′AAA→GCA BsaAI
R147A5′-CCTTATGTATACAAAGCAAACAATG-3′AGA→GCA AccI
D150A5′-GAAACAATGCATGGGTGGTT-3′GAC→GCA EcoT22I
H157Af 5′-TGTGGTGTAGCTGCAGCTGC-3′CAT→GCT PstI
H157A-2f 5′-TTGTGGTGTAGCCGCGGCTGCCACGA-3′CAT→GCC SacII
H157Y5′-TTGTGGTGTATACGCAGCTGCCA-3′CAT→TAC AccI
H157Q5′-GTGGTGTACAGGCAGCTGCCA-3′CAT→CAG(NspI)d
H157R5′-TGTGGTGTACGTGCAGCTGCC-3′CAT→CGT BsaAI
K162A5′-GCAGCTGCCACGGCTAGCGGTAACACTGTA-3′AAG→GCT NheI
E175A5′-GTTCAGGCTGGCGCCGGTGAAACCAC-3′GAA→GCC EheI
E177A5′-GGGGAAGGCGCCACCACCCT-3′GAA→GCC EheI
E181A5′-GAAACCACGCTAGCGTAAGCAT-3′GAG→GCG NheI
  • a The name of the primer represents the amino acid change. Amino acids are shown in the one-letter code. Letters before the number indicate the original amino acid residues, and letters after the number indicate the introduced amino acid residues.

  • b Restriction sites used for detection of mutations are underlined.

  • c Since the BspMI and PshAI sites were not cleaved, R8Q, H30Q, H30N, and H30E mutations were detected by DNA sequencing.

  • d Restriction enzymes in parentheses indicate the enzymes that disappeared by mutagenesis.

  • e C139A was described as C1235A in reference 40.

  • f With the H157A primer, the Ser (CAT→TCT) and Pro (CAT→CCT) mutants were obtained instead of the Ala mutant. Therefore, the H157A-2 primer was used for the Ala mutation.