从大肠杆菌细胞中原位再生和提取重组水母发光蛋白以及提取的水母发光蛋白的纯化。
The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.
作者信息
Shimomura O, Inouye S
机构信息
Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
出版信息
Protein Expr Purif. 1999 Jun;16(1):91-5. doi: 10.1006/prep.1999.1049.
Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.
在大肠杆菌细胞周质空间中表达的重组脱辅基水母发光蛋白，在含有腔肠素的缓冲液作用下，同时再生为水母发光蛋白并从细胞中提取出来。由于提取条件温和，提取物中的杂质极少。因此，仅通过两步，即阴离子交换色谱法和疏水相互作用色谱法，简单地在两步中进行吸附和洗脱，就可以完成提取的水母发光蛋白的纯化。根据包括高效液相色谱分析和发光活性在内的各种数据，纯化后的重组水母发光蛋白是纯的。从600毫升培养物中纯化得到的水母发光蛋白产量为25 - 35毫克，这超过了大肠杆菌细胞中表达的脱辅基水母发光蛋白总量的75%。
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