FIG. 1.

Effect of replacement of cysteine residues on the production of A subunit. The cysteine residues at positions 187 and 199 were replaced by oligonucleotide-directed site-specific mutagenesis, and the gene fragments containing the mutant A-subunit gene were cloned into pET11 for selective expression as described in the text. E. coli BL21 cells were transformed with these mutant plasmids (lanes 2 and 3), wild-type plasmid (lane 1), and vector plasmid (lane 4). These transformed cells were induced with IPTG and labeled for 1 min with [³⁵S]methionine. The incubation continued for 30 s. The cells were collected by centrifugation, and the periplasmic fractions were prepared as described in the text. The periplasmic fractions were mixed with SDS dye solution, heated at 100°C for 10 min, and resolved by SDS-PAGE. Radioactive bands were detected by image analysis as described in the text. The arrow indicates the position of the A subunit.