FIG. 1.

Strategies for construction of the secretion vectors pSE420-93CTPB and pSE420-181CTPB and hybrid proteins and location of glycine-rich motifs in E. chrysanthemi protease B structure. (A) Relevant restriction endonuclease sites on the carboxyl terminus of the prtB gene cloned in pRUW500 and three strategies for construction of secretion vectors and hybrid genes are shown. The restriction fragment (a) corresponds to the 93CTPB coding region and was initially ligated into the EcoRV and SacII sites of pSE280 and then transferred to pSE420. The 181CTPB fragments (b and c) were obtained by PCR amplification as explained in the text. Relevant plasmid constructions are boxed. Details of hybrid constructions are given in Materials and Methods. The SmaI site shown in parentheses at the protease B gene was created by PCR to allow the in-frame ligation required for hybrid constructions. (B) Glycine-rich and hydrophobic motifs are displayed in the CTPB region. The vertical and diagonal arrows indicate the relative positions of the motifs (boxed sequences) along the amino acid protease B sequence. Numbers on the protease B graphic representation indicate amino acid positions. The 93- and 181-CTPB regions are shown as stippled boxes.