核苷酸序列,克隆和表达flavodoxin基因的脱磷孤菌属寻常的(Hildenborough)。
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克雷GD Vanin EF,斯文森RP
核苷酸序列,克隆和表达flavodoxin基因的脱磷孤菌属寻常的(Hildenborough)。
J生物化学杂志。1988年10月25日,263 (30):15436 - 43。
- PubMed ID
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3170590 (在PubMed]
- 文摘
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flavodoxin基因编码的蛋白质脱磷孤菌属寻常的(Hildenborough)已被确定,克隆和测序。DNA片段含有flavodoxin基因被确定由混合合成heptadecanucleotide探针的杂交南方滴SalI-digested基因组DNA。调查的核苷酸序列来自出版蛋白质一级结构(Dubourdieu, M。LeGall, J。学生物化学,福克斯,j·l . (1973)。Biophys。Commun >, 52岁,1418 - 1425)。相同的寡核苷酸探针用于屏幕库(pUC19)包含size-selected萨利·碎片。重组,携带1.6千碱基(kb)插入强烈与探针,被发现含有核苷酸序列编码的前104残留的伴部分flavodoxin蛋白质序列但缺乏剩余的基因。因此,PstI限制从这个克隆片段作为探针隔离整个基因部分Sau3AI图书馆摆渡的船夫35。强烈的斑块继续杂交探针通过重复放映,重组,包含16 kb插入,进一步为特征。 The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.