生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 90-98.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0129
李奕雅1(), 吴一凡1, 丁能水2, 范小萍2, 陈凡1()
收稿日期:
2023-02-15
出版日期:
2023-12-26
发布日期:
2024-01-11
通讯作者:
陈凡,男,博士,副教授,研究方向:细胞生物学;E-mail: cf@bio.mnnu.edu.cn作者简介:
李奕雅,女,硕士研究生,研究方向:蛋白质功能与应用;E-mail: lyy@bio.mnnu.edu.cn
基金资助:
LI Yi-ya1(), WU Yi-fan1, DING Neng-shui2, FAN Xiao-ping2, CHEN Fan1()
Received:
2023-02-15
Published:
2023-12-26
Online:
2024-01-11
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。
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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