NUCLEOTIDE-SEQUENCE OF THE STRUCTURAL GENE FOR DIPHTHERIA-TOXIN CARRIED BY CORYNEBACTERIOPHAGE-BETA
NUCLEOTIDE-SEQUENCE OF THE STRUCTURAL GENE FOR DIPHTHERIA-TOXIN CARRIED BY CORYNEBACTERIOPHAGE-BETA
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DOI：
10.1073/pnas.80.22.6853
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
KAPLAN, DA
中科院分区：
文献类型：
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作者：
GREENFIELD, L;BJORN, MJ;KAPLAN, DA
对编码白喉毒素结构基因的1942个碱基对的DNA片段进行了测序，推测了白喉毒素的一级结构。从棒状噬菌体β中分离出与毒素的无毒或低毒多肽相对应的限制性内切酶片段。将其克隆到pBR322载体上，并进行序列测定。由该序列推断的成熟毒素分子含有氨基酸残基，分子量为58,342。推导出的片段A部分的序列与在蛋白质水平上确定的序列相同，除了一个丝氨酸残基，这在早期的研究中被错定位了。关于片段B部分的部分序列数据，注意到了几个不同之处，其中一些或全部可能反映了携带毒素基因的肾小球虫种群之间的遗传差异。DNA序列预测在成熟蛋白之前有一个25个残基的前导肽，这可能参与了溶原化白喉棒状杆菌毒素的分泌。显然，翻译的起始可能发生在GTG密码子(密码子-25)。克隆了含有毒素氨基末端及其5‘端序列的限制性内切酶片段，并在大肠杆菌中进行了表达。毒素相关多肽被合成并分泌到周质间隙。这些结果为将重组DNA方法应用于白喉毒素的研究和生产适合构建新型免疫毒素的新型、经基因改造的白喉毒素奠定了基础。
A 1942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage .beta. and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a MW of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. Evidently, initiation of the translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5'' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.