荧光素酶辅助定量大肠杆菌破碎效果的方法

doi:10.13560/j.cnki.biotech.bull.1985.2023-0129

摘要/Abstract

摘要：

建立基于萤火虫荧光素酶的大肠杆菌超声破碎辅助定量方法，利用荧光素酶灵敏度高、检测迅速的特点，对靶细胞的破碎效果进行表征。以表达了萤火虫荧光素酶的大肠杆菌作为内标，在破碎前与靶蛋白表达菌按一定比例混合，考察不同破碎条件下荧光素酶和靶蛋白活性向胞外释放的情况，对辅助定量效果进行评价。结果表明，将表达了萤火虫荧光素酶的大肠杆菌按1∶500（体积比）同待破碎菌悬液混合，能够通过破碎后上清液中荧光素酶活性的变化，正确反映靶细胞的破碎程度，并为破碎过程中蛋白质的活性保存提供参考。破碎产物中引入的荧光素酶，对后续靶蛋白的镍珠法纯化没有影响。含有萤火虫荧光素酶的菌体，可在-80℃环境下保存至少90 d而不产生酶活性的显著下降，且细胞对超声破碎的耐受性也不因冻存改变，可在临用前直接解冻并与靶细胞混合，起到辅助定量作用。因此，利用萤火虫荧光素酶对大肠杆菌超声破碎的程度进行辅助定量，是简便、稳定且高效的。

关键词: 荧光素酶, 超声破碎, 大肠杆菌, 定量分析

Abstract:

In this study, we present a novel and effective method for measuring ultrasonic disruption of Escherichia coli cells based on firefly luciferase. This quantitative approach provides high sensitivity and rapid detection of cell disruption, by which the disruption was characterized. To evaluate this method, we employed an E. coli strain expressing firefly luciferase as an internal reference, which was mixed with the target bacterial suspension at a specific ratio before sonication. The release of both luciferase and target protein activities into the extracellular solution under different ultrasonic conditions was then investigated, and the assisted quantitative effect was evaluated. The results demonstrated that: A ratio of 1∶500（volume ratio）E. coli strain expressing firefly luciferase added to the target bacterial suspension was sufficient for sonication quantification. The effect of sonication was calculated by the change of luciferase activity within the supernatant. Importantly, luciferase activity also provided clues for maintaining target protein activity during sonication. The luciferase protein incorporated into the final supernatant showed no effect on the purification of the target protein by nickel beads. Bacteria containing firefly luciferase can be stored at -80℃ for at least 90 d without significant loss of luciferase activity. Moreover, the disruption resistance was not changed during the long-term cryo-storage process. Therefore, freezing bacterial suspension can be thawed and directly mixed with target cells for assisting quantification without losing accuracy. In conclusion, the luciferase-assisted quantification of E. coli ultrasonic disruption is convenient, robust, and efficient.

Key words: luciferase, ultrasonic disruption, Escherichia coli, quantitative analysis

李奕雅, 吴一凡, 丁能水, 范小萍, 陈凡. 荧光素酶辅助定量大肠杆菌破碎效果的方法[J]. 生物技术通报, 2023, 39(12): 90-98.

LI Yi-ya, WU Yi-fan, DING Neng-shui, FAN Xiao-ping, CHEN Fan. Establishment of a Luciferase-assisted Quantitative Method for Measuring Ultrasonic Disruption of Escherichia coli Cells[J]. Biotechnology Bulletin, 2023, 39(12): 90-98.

图/表 7

图1 本研究使用的载体结构示意图 A：在pCold III DNA载体翻译增强子下游插入萤火虫或海肾荧光素酶CDS（coding sequences），二者均不携带亲和纯化标签；B：在pCold III DNA载体翻译增强子下游插入EGFP或mCherry CDS，二者C端均携带组氨酸亲和纯化标签（His-Tag）。所构建的载体中，cspA启动子（cspA promoter）的作用受乳糖操纵序列的抑制，向培养基中添加IPTG（isopropyl β-D-thiogalactoside）后，抑制被解除，目的基因得到表达

Fig. 1 Schematic representation of the plasmid structures used in this study A: Firefly or Renilla luciferase CDS was inserted into the downstream of the translation enhancing element of pCold III DNA plasmids without affinity tags for purification;B: EGFP or mCherry CDS was inserted into the downstream of the translation enhancing element of pCold III DNA plasmids with a C-terminal His-Tag for purification. Among the functional elements of each plasmid, the function of cspA promoter is inhibited by lac operator and the inhibition is released after IPTG（isopropyl β-D-thiogalactoside）is added, and target gene is expressed

图2 经诱导表达的4种蛋白质活性标准曲线

Fig. 2 Standard curves of four induced proteins activities after induction

图3 三种菌悬液超声破碎的萤火虫荧光素酶辅助定量结果图以破碎样品中活性最强的平均值为100%，计算菌体破碎程度

Fig. 3 Firefly luciferase-assisted quantification of ultrasonic disruption for three bacterial suspensions The degree of body fragmentation was calculated using the mean value of the strongest activity in the disrupted sample as 100%

图4 不同处理温度下4种蛋白质活性的变化各图中以0℃处理样品的活性为100%，无相同小写字母的两组差异显著（P<0.05）

Fig. 4 Activity changes of four proteins incubated under different temperatures The remaining sample activity incubated under 0℃ in each plot was considered 100%. Groups do not share any lower letter indicate significant difference（P<0.05）

图5 不同超声破碎功率对破碎后荧光素酶活性的影响各图中FLuc和RLuc对应的最大读数均值（100%活性）已标注在图中

Fig. 5 Effects of different ultrasonic power on luciferase activity after sonication The means of maximum readings（100% activity）corresponding to FLuc and RLuc are labeled in each plot

图6 萤火虫荧光素酶的添加对靶蛋白后续纯化的影响 A：EGFP表达菌破碎上清液的梯度荧光照片；B：mCherry表达菌破碎上清液的梯度荧光照片；C：FLuc表达菌破碎上清液的梯度荧光照片；D：镍珠纯化前后混合样品的SDS-PAGE电泳图谱

Fig. 6 Effect of firefly luciferase addition on the purification of target proteins A: Gradient fluorescence intensity graph of EGFP-expressing bacterial supernatant sample after sonication. B: Gradient fluorescence intensity graph of mCherry-expressing bacterial supernatant sample after sonication. C: Gradient fluorescence intensity graph of FLuc-expressing bacterial supernatant sample after sonication. D: SDS-PAGE image of mixed samples before and after nickel bead purification

图7 不同冻存时间对萤火虫荧光素酶辅助定量效果的影响每条曲线后3个数据点的平均值作为菌体100%破碎的指标已直接标注于图中，且每条曲线3 min处所对应数据点的平均值和该数值对应的破碎率也已标注

Fig. 7 Effect of different cryo-storage time on firefly luciferase-assisted quantification For each curve, the mean readings at 15, 18, and 21 min were averaged and considered as the value（labeled directly in each figure）indicating a complete disruption of specific bacterial cells. The mean reading at 3 min and its ratio against complete disruption were also marked for comparison

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