CLONING OF THE LUCIFERASE STRUCTURAL GENES FROM VIBRIO-HARVEYI AND EXPRESSION OF BIOLUMINESCENCE IN ESCHERICHIA-COLI
CLONING OF THE LUCIFERASE STRUCTURAL GENES FROM VIBRIO-HARVEYI AND EXPRESSION OF BIOLUMINESCENCE IN ESCHERICHIA-COLI
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DOI：
10.1021/bi00311a014
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
ZIEGLER, MM
中科院分区：
文献类型：
--
作者：
BALDWIN, TO;BERENDS, T;ZIEGLER, MM
编码荧光素酶α的DNA。和β发光海洋细菌哈维氏弧菌（菌株392）中的亚基包含在4.0-内切酶HindIII片段内。用HindIII消化来自哈维氏弧菌的DNA，将所得片段插入质粒pBR 322的HindIII位点。将重组质粒转化E. coli RR1。向菌落提供正癸醛（生物发光反应的底物），并将12个菌落（约共6000个）被观察到明亮地发光。对其中一个重组质粒pTB 7进行了详细的研究。pTB 7中生物发光的高水平表达不是天然哈氏弧菌启动子的结果，而是pBR 322中的启动子的结果，该启动子在四环素抗性基因内，但取向与四环素基因的转录相反。用荧光素酶抗体检测十二烷基硫酸钠-聚丙烯酰胺凝胶转移到硝酸纤维素纸上的蛋白质，结果表明，E。大肠杆菌转化体产生与抗荧光素酶抗体交叉反应的荧光素酶，并且与哈维氏弧菌荧光素酶具有相同的MW。不是α。亚基可以通过在亚克隆pTB 104的裂解物中使用抗荧光素酶抗体来检测，pTB 104与pTB 7相同，除了β-亚基缺失之外。亚基基因因此，亚基可能是不稳定的并且被降解，除非它与β相关。本文报道了哈维氏弧菌（V.harveyi）和E.携带pTB 7的大肠杆菌转化体是不可区分的。在此基础上，我们得出结论：E.大肠杆菌能够为荧光素酶提供FMNH 2，并且生物发光不需要能量转移系统。
The DNA encoding the luciferase .alpha. and .beta. subunits in the luminous marine bacterium V. harveyi (strain 392) is contained within a 4.0-kilobase HindIII fragment. DNA from V. harveyi was digested with HindIII, and the resulting fragments were inserted into the HindIII site of plasmid pBR322. The recombinant plasmids were introduced by transformation into E. coli RR1. The colonies were supplied with n-decanal, the substrate for the bioluminescence reaction, and 12 colonies (of .apprx. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, was studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V. harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene. Using antiluciferase antibody to probe proteins transferred from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose paper, it was shown that the E. coli transformants produce luciferase that cross-reacts with antiluciferase antibody and is the same MW as V. harveyi luciferase. No .alpha. subunit could be detected by using antiluciferase antibody in lysates of a subclone, pTB104, which is identical with pTB7 except for deletion of the .beta.-subunit gene. Thus, the .alpha. subunit may be unstable and be degraded unless it is associated with .beta.. The bioluminescence emission spectra of V. harveyi and of E. coli transformants carrying pTB7 are indistinguishable. On the basis of these observations, it is concluded that enzyme(s) within E. coli is (are) capable of supplying luciferase with FMNH2 and that no energy transfer system is required for bioluminescence.