Cormier M J, Prasher D C, Longiaru M, McCann R O
Photochem Photobiol. 1989 Apr;49(4):509-12. doi: 10.1111/j.1751-1097.1989.tb09202.x.
Aequorin is a bioluminescent protein, isolated from the hydromedusan Aequorea victoria. A recombinant cDNA plasmid (pAEQ1) was shown to encode apoaequorin by detecting photoprotein activity in an extract of an E. coli strain containing pAEQ1 (Prasher et al., 1986, Biochem. Biophys. Res. Comm. 126, 1259-1268). The nucleotide sequence of the pAEQ1 insert has been determined and is shown to differ significantly from the aequorin cDNA (AQ440) isolated by Inouye et al. (1985, Proc. Natl. Acad. Sci. USA 82, 3154-3158). Comparisons of the coding regions of the two cDNAs show there are 52 nucleotide differences, 19 of which are responsible for 18 amino acid replacements. These differences explain the microheterogeneity observed at 17 positions during the sequencing of native apoaequorin. Five aequorin isotypes extracted from Aequorea tissue are observed on 2-dimensional gels and the E. coli-expressed apoaequorin is shown to co-migrate with one of these isotypes. The multiple isotypes could be caused by the presence of a multi-gene family since Southern blot analysis of Aequorea DNA suggests the presence of a minimum of four aequorin genes. Immunoblot analysis suggests that purified native aequorin is proteolytically cleaved during its purification from Aequorea. Comparison of the deduced cDNA translations and the protein sequence suggests the loss of seven residues from the amino terminal. Overexpression of the apoaequorin cDNA in E. coli now provides the means of obtaining gram quantities of a single isotype of the protein which can be converted to aequorin in the presence of coelenterate luciferin, oxygen and an appropriate thiol. Proper extraction procedures and a single chromatographic step provides apoaequorin which is greater than 95% homogeneous.
水母发光蛋白是一种生物发光蛋白，从水螅水母维多利亚多管水母中分离得到。通过检测含有pAEQ1的大肠杆菌菌株提取物中的光蛋白活性，证明了重组cDNA质粒（pAEQ1）编码脱辅基水母发光蛋白（普拉舍尔等人，1986年，《生物化学与生物物理学研究通讯》126卷，1259 - 1268页）。已确定pAEQ1插入片段的核苷酸序列，结果显示其与井上等人（1985年，《美国国家科学院院刊》82卷，3154 - 3158页）分离得到的水母发光蛋白cDNA（AQ440）有显著差异。对这两种cDNA编码区的比较表明，存在52个核苷酸差异，其中19个差异导致了18个氨基酸替换。这些差异解释了在天然脱辅基水母发光蛋白测序过程中在17个位置观察到的微异质性。在二维凝胶上观察到从多管水母组织中提取的五种水母发光蛋白同型异构体，并且大肠杆菌表达的脱辅基水母发光蛋白与其中一种同型异构体共迁移。多种同型异构体可能是由多基因家族的存在引起的，因为对多管水母DNA的Southern印迹分析表明至少存在四个水母发光蛋白基因。免疫印迹分析表明，从多管水母中纯化天然水母发光蛋白的过程中，它会被蛋白酶切割。推导的cDNA翻译产物与蛋白质序列的比较表明，氨基末端缺失了七个残基。在大肠杆菌中脱辅基水母发光蛋白cDNA的过表达现在提供了获得克量级单一同型异构体蛋白质的方法，该蛋白质在存在腔肠动物荧光素、氧气和适当硫醇的情况下可转化为水母发光蛋白。适当的提取程序和单一色谱步骤可提供纯度大于95%的脱辅基水母发光蛋白。
