FIG. 1.

Construction and expression analysis of CTB genes in P. parstoris. (A) A schematic representation of five CTB gene constructs: pB, a full-length CTB gene (375-bp open reading frame) containing a 21-amino-acid original leader sequence; psB, P. pastoris codon-optimized full-length synthetic CTB gene; pmB, matured form of CTB gene devoid of the leader sequence but provided with the initiation codon immediately in front of the first amino acid residue of the matured CTB protein; pB^ER, the full-length CTB gene containing a tetrapeptide yeast microsomal retention signal (HDEL) at the C terminus; pαB, matured form of CTB gene fused in frame with the S. cerevisiae alpha-factor secretion signal at the N terminus. (B) Intra- and extracellular CTB pentamer expressions were analyzed by GM1-ELISA from cellular extract and culture supernatant, respectively. For cellular extract, 1 ml of sample was prepared from approximately 1.2 × 10⁸ cells, determined by the OD₆₀₀. Estimated percent levels of intracellular CTB pentamer proteins in yeast total soluble protein are provided. Data are representative of a clone with the highest expression level screened from up to 50 independent transformants. The lower panel shows secreted expression of CTB pentamer proteins detected by the Western blotting of crude culture supernatant. aa, amino acids; ND, not determined.