Nakayama A, Shimada H, Furutani Y, Honjo M
Biotechnology Laboratory, Mitsui Toatsu Chemicals, Inc., Mobara, Japan.
J Biotechnol. 1992 Mar;23(1):55-69. doi: 10.1016/0168-1656(92)90099-u.
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.
构建了一组解淀粉芽孢杆菌中性蛋白酶基因前原肽的嵌套3'端缺失体。利用这些截短基因在枯草芽孢杆菌中分泌α-淀粉酶和人生长激素。分泌的α-淀粉酶水平随融合基因中截短前原肽的区域而变化，但与它的长度无关。尽管前原肽的长度不同，但分泌的α-淀粉酶的迁移率与来自α-淀粉酶克隆pTUB4（Yamazaki等人，1983）的对照α-淀粉酶相同。对分泌的N端氨基酸序列的分析证实它们都与天然的相同。在细胞相关部分发现了α-淀粉酶的前体蛋白，这表明前原肽部分在分泌过程中被加工。另一方面，使用这些前原肽部分之一分泌的hGH的N端因来自前肽部分与成熟hGH序列之间的连接序列或前肽部分的C端区域的1至4个额外的N端氨基酸残基而有所不同。这些结果表明，即使在异源融合中，前原肽部分通常也能被加工。还讨论了融合基因产物加工和成熟的可能机制。
