Establishment of a Luciferase-assisted Quantitative Method for Measuring Ultrasonic Disruption of Escherichia coli Cells

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

Abstract

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

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.

Figures/Tables 7

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

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

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%

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）

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

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

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

References 27

[1]	张磊, 唐永凯, 李红霞, 等. 促进原核表达蛋白可溶性的研究进展[J]. 中国生物工程杂志, 2021, 41(S1): 138-149.
	Zhang L, Tang YK, Li HX, et al. Advances in promoting solubility of prokaryotic expressed proteins[J]. China Biotechnol, 2021, 41(S1): 138-149.
[2]	Li SY, Zuo DY, Cheng HL, et al. Glutathione S-transferases GhGSTF1 and GhGSTF2 involved in the anthocyanin accumulation in Gossypium hirsutum L[J]. Int J Biol Macromol, 2020, 165(Pt B): 2565-2575. doi: 10.1016/j.ijbiomac.2020.10.101 URL
[3]	Bai YJ, Guo JR, Reiter RJ, et al. Melatonin synthesis enzymes interact with ascorbate peroxidase to protect against oxidative stress in cassava[J]. J Exp Bot, 2020, 71(18): 5645-5655. doi: 10.1093/jxb/eraa267 pmid: 32474586
[4]	李航, 戚睿斌, 陈宗艳, 等. 外源蛋白表达系统及其应用的研究进展[J]. 黑龙江畜牧兽医, 2021(7): 34-37, 47.
	Li H, Qi RB, Chen ZY, et al. Progress in research on foreign protein expression system and its application[J]. Heilongjiang Anim Sci Vet Med, 2021(7): 34-37, 47.
[5]	Martins M, Ooi CW, Neves MC, et al. Extraction of recombinant proteins from Escherichia coli by cell disruption with aqueous solutions of surface-active compounds[J]. J Chem Technol Biotechnol, 2018, 93(7): 1864-1870.
[6]	游思亮, 王裔娜, 田瑞平, 等. 响应面法优化高压均质破碎重组大肠杆菌的条件[J]. 江苏农业科学, 2021, 49(17): 71-74.
	You SL, Wang YN, Tian RP, et al. Optimization of high-pressure homogeneous disruption of recombinant Escherichia coli by response surface methodology[J]. Jiangsu Agric Sci, 2021, 49(17): 71-74.
[7]	吴蕾, 雷鸣, 洪建辉, 等. 超声破碎重组大肠杆菌释放包含体的过程研究[J]. 化学工业与工程, 2002, 19(6): 422-425.
	Wu L, Lei M, Hong JH, et al. Studies of ultrasonic disruption process to release inclusion body in recombinant E. coli[J]. Chem Ind Eng, 2002, 19(6): 422-425. doi: 10.1021/ie50207a031 URL
[8]	Falgenhauer E, von Schönberg S, Meng C, et al. Evaluation of an E. coli cell extract prepared by lysozyme-assisted sonication via gene expression, phage assembly and proteomics[J]. Chembiochem, 2021, 22(18): 2805-2813. doi: 10.1002/cbic.202100257 pmid: 34240805
[9]	Kuduğ H, Ataman B, İmamoğlu R, et al. Production of red fluorescent protein(mCherry)in an inducible E. coli expression system in a bioreactor, purification and characterization[J]. Int Adv Res Eng J, 2019(1): 20-25.
[10]	Branchini BR, Southworth TL, Fontaine DM, et al. A firefly luciferase dual color bioluminescence reporter assay using two substrates to simultaneously monitor two gene expression events[J]. Sci Rep, 2018, 8(1): 5990. doi: 10.1038/s41598-018-24278-2 pmid: 29662072
[11]	薛秀花, 黄晨西. 荧光素酶基因的研究进展[J]. 生物学通报, 2013, 48(9): 1-4.
	Xue XH, Huang CX. Research progress of luciferase gene[J]. Bull Biol, 2013, 48(9): 1-4.
[12]	Close DM, Xu TT, Sayler GS, et al. In vivo bioluminescent imaging(BLI): noninvasive visualization and interrogation of biological processes in living animals[J]. Sensors, 2011, 11(1): 180-206. doi: 10.3390/s110100180 pmid: 22346573
[13]	Zhang YQ, Dai YP, Wang JX, et al. Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity[J]. EMBO J, 2022, 41(11): e109902. doi: 10.15252/embj.2021109902 URL
[14]	Amadeo F, Plagge A, Chacko A, et al. Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies[J]. Eur J Nucl Med Mol Imaging, 2022, 49(3): 796-808. doi: 10.1007/s00259-021-05439-4
[15]	Saito-Moriya R, Nakayama J, Kamiya G, et al. How to select firefly luciferin analogues for in vivo imaging[J]. Int J Mol Sci, 2021, 22(4): 1848. doi: 10.3390/ijms22041848 URL
[16]	Ihssen J, Jovanovic N, Sirec T, et al. Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase[J]. PLoS One, 2021, 16(1): e0244200. doi: 10.1371/journal.pone.0244200 URL
[17]	Pozzo T, Akter F, Nomura Y, et al. Firefly luciferase mutant with enhanced activity and thermostability[J]. ACS Omega, 2018, 3(3): 2628-2633. doi: 10.1021/acsomega.7b02068 pmid: 30023842
[18]	Bjerga GEK, Williamson AK. Cold shock induction of recombinant Arctic environmental genes[J]. BMC Biotechnol, 2015, 15: 78. doi: 10.1186/s12896-015-0185-1 pmid: 26286037
[19]	Duellman T, Burnett J, Yang J. Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader[J]. Anal Biochem, 2015, 473: 34-40. doi: 10.1016/j.ab.2014.12.010 pmid: 25542417
[20]	Rahman MM, Lamsal BP. Ultrasound-assisted extraction and modification of plant-based proteins: impact on physicochemical, functional, and nutritional properties[J]. Compr Rev Food Sci Food Saf, 2021, 20(2): 1457-1480. doi: 10.1111/crf3.v20.2 URL
[21]	Ohmuro-Matsuyama Y, Gomi K, Shimoda T, et al. Improving the stability of protein-protein interaction assay FlimPIA using a thermostabilized firefly luciferase[J]. Front Bioeng Biotechnol, 2021, 9: 778120. doi: 10.3389/fbioe.2021.778120 URL
[22]	Koksharov MI, Ugarova NN. Thermostabilization of firefly luciferase by in vivo directed evolution[J]. Protein Eng Des Sel, 2011, 24(11): 835-844. doi: 10.1093/protein/gzr044 pmid: 21900306
[23]	Zhou FF, Zhang L, Gong K, et al. LEF-1 activates the transcription of E2F1[J]. Biochem Biophys Res Commun, 2008, 365(1): 149-153. doi: 10.1016/j.bbrc.2007.10.138 URL
[24]	Li TT, Kong AN T, Ma ZQ, et al. Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant[J]. Oncotarget, 2016, 7(15): 20236-20248. doi: 10.18632/oncotarget.7752 pmid: 26934120
[25]	Yang L, Chen Y, Liu N, et al. CircMET promotes tumor proliferation by enhancing CDKN2A mRNA decay and upregulating SMAD3[J]. Mol Cancer, 2022, 21(1): 23. doi: 10.1186/s12943-022-01497-w pmid: 35042525
[26]	Simpson RJ. Stabilization of proteins for storage[J]. Cold Spring Harb Protoc, 2010, 2010(5): 1-14.
[27]	Golovanov AP, Hautbergue GM, Wilson SA, et al. A simple method for improving protein solubility and long-term stability[J]. J Am Chem Soc, 2004, 126(29): 8933-8939. doi: 10.1021/ja049297h pmid: 15264823