Kadonaga J T, Gautier A E, Straus D R, Charles A D, Edge M D, Knowles J R
J Biol Chem. 1984 Feb 25;259(4):2149-54.
A derivative of pBR322 has been constructed that contains both a unique EcoRI restriction site right at the beginning of the signal codons of the beta-lactamase (bla) gene and a unique BstEII site just at the end of the bla signal codons. Although the signal peptide encoded by the new plasmid differs from the wild type (pBR322) by 2 amino acid residues (Ser 2 to Arg 2 and Ala 23 to Gly 23), the synthesis, transport, and processing of the beta-lactamase remain unchanged in Escherichia coli. Two deletion mutants, in which the bla signal codons have been almost completely excised, have also been constructed. Bacteria containing either of these plasmids produce, but do not secrete, an active beta-lactamase. Last, the bla signal codons have been precisely joined to the cDNA version of the triose phosphate isomerase (tpi) gene from chicken. Expression of this fusion gene in E. coli gives a hybrid protein that is neither secreted into the periplasm nor proteolytically processed. This result supports the view that there are characteristics of the mature protein that are necessary for the secretion across the inner membrane of E. coli.
构建了一种pBR322的衍生物，它在β-内酰胺酶（bla）基因信号密码子起始位置正好有一个独特的EcoRI限制性酶切位点，且在bla信号密码子末端正好有一个独特的BstEII位点。尽管新质粒编码的信号肽与野生型（pBR322）相比有2个氨基酸残基不同（Ser2变为Arg2以及Ala23变为Gly23），但β-内酰胺酶的合成、转运和加工在大肠杆菌中保持不变。还构建了两个缺失突变体，其中bla信号密码子几乎被完全切除。含有这两种质粒中任何一种的细菌都会产生但不分泌有活性的β-内酰胺酶。最后，bla信号密码子已精确连接到来自鸡的磷酸丙糖异构酶（tpi）基因的cDNA版本。该融合基因在大肠杆菌中的表达产生一种既不分泌到周质中也不进行蛋白水解加工的杂合蛋白。这一结果支持了这样一种观点，即成熟蛋白具有一些对于穿过大肠杆菌内膜进行分泌所必需的特征。
