Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein.
The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp. carotovora has been cloned previously [Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044]. We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens. To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions. Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence. There was approximately fourfold variation in the production efficiency of B. subtilis strains carrying the different constructs. The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide. This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium. In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs. Our results suggest that fusions containing intact signal peptides, which are common when producing foreign proteins, are not necessarily the most efficient.
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Institute of Biotechnology, University of Helsinki, Finland.
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D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases; D011096:Polygalacturonase