CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION IN ESCHERICHIA-COLI OF THE EXOTOXIN-A STRUCTURAL GENE OF PSEUDOMONAS-AERUGINOSA
CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION IN ESCHERICHIA-COLI OF THE EXOTOXIN-A STRUCTURAL GENE OF PSEUDOMONAS-AERUGINOSA
复制标题
DOI：
10.1073/pnas.81.9.2645
复制
发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
HEYNEKER, HL
中科院分区：
文献类型：
--
作者：
GRAY, GL;SMITH, DH;HEYNEKER, HL
将编码外毒素 A (ETA) 结构基因的铜绿假单胞菌菌株 PA103 染色体的 2760 个碱基对 DNA 片段克隆到大肠杆菌中，并测定了核苷酸序列。对 5'' 和 3'' 侧翼区域的分析表明 ETA 是从单顺反子信息翻译而来的。将推导的 NH2 末端氨基酸序列与通过分泌蛋白的序列分析确定的序列进行比较表明，ETA 是由 638 个氨基酸组成的前体，在分泌过程中，25 个氨基酸的高度疏水性前导肽被去除。所提供的数据表明该分子的 ADP-核糖基化活性的 COOH 末端位置。 ETA编码序列在大肠杆菌trp启动子而非ETA启动子控制下在大肠杆菌中表达，导致酶活性和免疫活性蛋白质的产生。大多数这种物质似乎既没有被加工也没有被分泌。将ETA氨基酸和核苷酸序列与白喉毒素的氨基酸和核苷酸序列进行比较，发现没有显着的同源性，表明这些功能相似的毒素是从不同的基因进化而来的。
A 2760-base pair DNA segment of the P. aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene was cloned in E. coli and the nucleotide sequence was determined. Analysis of the 5''- and 3''-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. Most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.