Ladant D, Glaser P, Ullmann A
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
J Biol Chem. 1992 Feb 5;267(4):2244-50.
We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.
我们开发了一种改进的接头插入诱变方法，用于将2个或16个密码子引入百日咳博德特氏菌的cyaA基因，该基因编码一种钙调蛋白依赖性腺苷酸环化酶。一个含有寡核苷酸接头的重组卡那霉素抗性盒被克隆到携带截短的cyaA基因的质粒中，该基因在其3'端与大肠杆菌lacZ基因的5'端融合，编码α-肽。这种构建允许通过筛选卡那霉素抗性和α-互补产生的乳糖阳性表型对框内插入进行双重选择。我们发现，腺苷酸环化酶催化结构域N端部分的大多数两氨基酸插入会消除酶活性和/或改变蛋白质的稳定性。腺苷酸环化酶C端部分的所有两氨基酸插入都产生了完全稳定且有活性的酶。这些结果证实了先前基于蛋白水解研究提出的腺苷酸环化酶催化结构域的模块化结构。在残基247 - 248和335 - 336之间的两氨基酸插入被证明会影响腺苷酸环化酶对钙调蛋白的反应性，表明该酶中的相应区域参与钙调蛋白的结合或钙调蛋白激活过程。此外，我们在腺苷酸环化酶的一级结构中确定了几个允许位点，这些位点可耐受16个氨基酸的插入而不干扰催化活性或钙调蛋白结合。通过将外源抗原决定簇插入这些允许位点，所得的重组腺苷酸环化酶毒素可用于将特定表位递送至抗原呈递细胞。
