Processing of the prepropeptide portions of the Bacillus amyloliquefaciens neutral protease fused to Bacillus subtilis alpha-amylase and human growth hormone during secretion in Bacillus subtilis.
A set of nested 3'-terminal deletions of the prepropeptide of the Bacillus amyloliquefaciens neutral protease gene was constructed. Alpha-amylase and human growth hormone were secreted using these truncated genes in Bacillus subtilis. The level of the secreted alpha-amylase varied with the region for the truncated prepropeptide contained in the fusion gene but was independent of its length. Even though length of the prepropeptide varied, the mobilities of secreted alpha-amylases were the same as that of the control alpha-amylase derived from the alpha-amylase clone, pTUB4 (Yamazaki et al., 1983). Analyses of the secreted N-terminal amino acid sequences confirmed that they were all identical to that of the authentic one. Precursor proteins of the alpha-amylase were found in the cell-associated fraction, suggesting that the prepropeptide portion was processed during secretion. On the other hand, the N-terminus of hGH secreted using one of these prepropeptide portions varied by 1 to 4 additional N-terminal amino acid residues derived from the junction sequence between the sequence for propeptide portion and mature hGH or from C-terminal region of the propeptide portion. These results suggest that the prepropeptide portion can be generally processed even in the heterogeneous fusion. A probable mechanism of processing and maturation of the fusion gene products is also discussed.
Biotechnology Laboratory, Mitsui Toatsu Chemicals, Inc., Mobara, Japan.
A
A
Nakayama
H
H
Shimada
Y
Y
Furutani
M
M
Honjo
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002458:Cell Fractionation; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics*; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D000516:alpha-Amylases; D010450:Endopeptidases