Lingappa V R, Chaidez J, Yost C S, Hedgpeth J
Proc Natl Acad Sci U S A. 1984 Jan;81(2):456-60. doi: 10.1073/pnas.81.2.456.
A hybrid gene containing 182 codons of Escherichia coli beta-lactamase at the amino terminus of the corresponding protein and 141 codons of alpha-globin at the carboxyl terminus was generated by inserting chimpanzee alpha-globin cDNA into the Pst I site of plasmid pBR322. RNA transcribed in vitro from this plasmid gave a corresponding hybrid protein in a wheat germ cell-free translation system. The hybrid protein was protected from tryptic digestion and the pre-beta-lactamase signal peptide was removed when dog pancreas membrane vesicles were present during translation. A deletion mutant containing 23 codons of pre-beta-lactamase signal sequence and 5 codons of mature beta-lactamase fused to the alpha-globin cDNA gave a shorter hybrid protein that behaved similarly. However, a mutation that removed essentially all of the pre-beta-lactamase sequence gave a protein that was neither protected nor processed. Hence, at most, only the signal peptide and the first 5 amino acids of beta-lactamase were necessary to convert alpha-globin (a cytoplasmic protein) into a secretory protein.
通过将黑猩猩α-珠蛋白cDNA插入质粒pBR322的Pst I位点，构建了一种杂合基因，该基因在相应蛋白质的氨基末端含有182个大肠杆菌β-内酰胺酶密码子，在羧基末端含有141个α-珠蛋白密码子。从该质粒体外转录的RNA在麦胚无细胞翻译系统中产生了相应的杂合蛋白。当翻译过程中存在犬胰腺膜泡时，杂合蛋白可免受胰蛋白酶消化，并且前β-内酰胺酶信号肽被去除。一个缺失突变体，其包含23个前β-内酰胺酶信号序列密码子和5个成熟β-内酰胺酶密码子与α-珠蛋白cDNA融合，产生了一种较短的杂合蛋白，其表现类似。然而，一个基本上去除了所有前β-内酰胺酶序列的突变产生了一种既不受保护也不被加工的蛋白质。因此，最多只有信号肽和β-内酰胺酶的前5个氨基酸对于将α-珠蛋白（一种细胞质蛋白）转化为分泌蛋白是必要的。
