Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.
A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.
Botes
D P
DP
Department of Biochemistry, University of Cape Town, Rondebosch, South Africa.
Qobose
M D
MD
Corfield
V A
VA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000235:genetics; D010957:Plasmids; D014361:Trypsin Inhibitors / Q000096:biosynthesis* / Q000235:genetics
D004277:DNA, Single-Stranded; D014361:Trypsin Inhibitors