Fluorescent Proteins as a Visible Molecular Signal for Rapid Quantification of Bioprocesses:Potential and Challenges

摘要/Abstract

摘要： Green fluorescent protein (GFP) and its variants/homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biological processes due to their capability of simple, accurate and real time quantification. The FPs-based molecular and visible quantification tools are giving more impact on bioprocess engineering, enabling the biomolecule-level dynamic information to be linked with the process-level events. In this review, different applications of FPs in biological engineering with emphasis on rapid molecular bioprocess quantification, such as quantification of the transcription efficiency, the protein production, the protein folding efficiency, the cell concentration, the intracellular microenvironments and so on, would be first introduced. The challenges of using FPs with respect to actual bioprocess applications for the precise quantification including the interaction of FPs and the fused partner proteins, the maturation of FPs, the inner filter effect and sensing technology were then discussed. Finally, the future development for the FPs used in molecular bioprocess quantification would be proposed.

关键词: green fluorescent protein, fluorescent proteins, bioprocess engineering, quantification, marker

Abstract: Green fluorescent protein (GFP) and its variants/homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biological processes due to their capability of simple, accurate and real time quantification. The FPs-based molecular and visible quantification tools are giving more impact on bioprocess engineering, enabling the biomolecule-level dynamic information to be linked with the process-level events. In this review, different applications of FPs in biological engineering with emphasis on rapid molecular bioprocess quantification, such as quantification of the transcription efficiency, the protein production, the protein folding efficiency, the cell concentration, the intracellular microenvironments and so on, would be first introduced. The challenges of using FPs with respect to actual bioprocess applications for the precise quantification including the interaction of FPs and the fused partner proteins, the maturation of FPs, the inner filter effect and sensing technology were then discussed. Finally, the future development for the FPs used in molecular bioprocess quantification would be proposed.

Key words: green fluorescent protein, fluorescent proteins, bioprocess engineering, quantification, marker

张翀, 邢新会. Fluorescent Proteins as a Visible Molecular Signal for Rapid Quantification of Bioprocesses:Potential and Challenges[J]. , 2010, 18(5): 863-869.

ZHANG Chong, XING Xinhui. Fluorescent Proteins as a Visible Molecular Signal for Rapid Quantification of Bioprocesses:Potential and Challenges[J]. , 2010, 18(5): 863-869.

参考文献

1 Shaner, N.C., Patterson, G.H., Davidson, M.W., “Advances in fluorescent protein technology”, J. Cell Sci., 120 (24), 4247-4260 (2007).
2 Stepanenko, O.V., Verkhusha, V.V., Kuznetsova, I.M., Uversky, V.N., Turoverov, K.K., “Fluorescent proteins as biomarkers and biosensors:Throwing color lights on molecular and cellular processes”, Curr. Protein. Pept. Sci., 9 (4), 338-369 (2008).
3 Davidson, M.W., Campbell, R.E., “Engineered fluorescent proteins:innovations and applications”, Nature Methods, 6 (10), 713-717 (2009).
4 Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N.G., Palmer, A.E., Tsien, R.Y., “Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein”, Nat. Biotechnol., 22 (12), 1567-1572 (2004).
5 March, J.C., Rao, G., Bentley, W.E., “Biotechnological applications of green fluorescent protein”, Appl. Microbiol. Biotechnol., 62 (4), 303-315 (2003).
6 Su, W.W., “Fluorescent proteins as tools to aid protein production”, Microb. Cell Fact., 4, 12 (2005).
7 Lu, C.H., Bentley, W.E., Rao, G., “A high-throughput approach to promoter study using green fluorescent protein”, Biotechnol. Progr., 20 (6), 1634-1640 (2004).
8 Kensy, F., Zang, E., Faulhammer, C., Tan, R.K., Buchs, J., “Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates”, Microb. Cell Fact., 8, 31 (2009).
9 Li, J.C., Wang, S., VanDusen, W.J., Schultz, L.D., George, H.A., Herber, W.K., Chae, H.J., Bentley, W.E., Rao, G., “Green fluorescent protein in Saccharomyces cerevisiae:Real-time studies of the GAL1 promoter”, Biotechnol. Bioeng., (2), 187-196 (2000).
10 Delvigne, F., Boxus, M., Ingels, S., Thonart, P., “Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli”, Microb. Cell Fact., 8, 15 (2009).
11 Fiorentino, G., Ronca, R., Bartolucci, S., “A novel E. coli biosensor for detecting aromatic aldehydes based on a responsive inducible archaeal promoter fused to the green fluorescent protein”, Appl. Microbiol. Biotechnol., 82 (1), 67-77 (2009).
12 Heneghan, M.N., Porta, C., Zhang, C., Burton, K.S., Challen, M.P., Bailey, A.M., Foster, G.D., “Characterization of serine proteinase expression in Agaricus bisporus and Coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 Promoter”, Appl. Environ. Microbiol., 75 (3), 792-801 (2009).
13 Chen, Y.P., Yan, J.A., Yang, M.J., Wang, J.W., Shen, D.L., “Expression of green fluorescent protein in Bacillus brevis under the control constitutive promoter F1 and insertion mutagenesis of F1 in Escherichia coli DH5 alpha”, FEMS Microbiol. Lett., 229 (1), 111-117 (2003).
14 Albano, C.R., Randers-Eichhorn, L., Bentley, W.E., Rao, G., “Green fluorescent protein as a real time quantitative reporter of heterologous protein production”, Biotechnol. Progr., 14 (2), 351-354 (1998).
15 Wu, C.F., Cha, H.J., Rao, G., Valdes, J.J., Bentley, W.E., “A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase”, Appl. Microbiol. Biotechnol., 54 (1), 78-83 (2000).
16 Chen, Y., Xing, X.H., Ye, F.C., Kuang, Y., “Soluble expression and rapid quantification of GFP-hepA fusion protein in recombinant Escherichia coli”, Chin. J. Chem. Eng., 15 (1), 122-126 (2007).
17 Niu, Y., Kong, J., Xu, Y., “A novel GFP-fused eukaryotic membrane protein expression system in Lactococcus lactis and its application to overexpression of an elongase”, Curr. Microbiol., 57 (5), 423-428 (2008).
18 Hammon, J., Palanivelu, D.V., Chen, J., Patel, C., Minor, D.L., “A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms”, Protein Science, 18 (1), 121-133 (2009).
19 Drew, D., Slotboom, D.J., Friso, G., Reda, T., Genevaux, P., Rapp, M., Meindl-Beinker, N.M., Lambert, W., Lerch, M., Daley, D.O., Van Wijk, K.J., Hirst, J., Kunji, E., De Gier, J.W., “A scalable, GFP-based pipeline for membrane protein overexpression screening and purification”, Protein Science, 14 (8), 2011-2017 (2005).
20 Wu, C.F., Cha, H.J., Valdes, J.J., Bentley, W.E., “GFP-visualized immobilized enzymes:Degradation of paraoxon via organophosphorus hydrolase in a packed column”, Biotechnol. Bioeng., 77 (2), 212-218 (2002).
21 Waldo, G.S., Standish, B.M., Berendzen, J., Terwilliger, T.C., “Rapid protein-folding assay using green fluorescent protein”, Nature Biotechnol., 17 (7), 691-695 (1999).
22 Vasiljevic, S., Ren, J.Y., Yao, Y.X., Dalton, K., Adamson, C.S., Jones, I.M., “Green fluorescent protein as a reporter of prion protein folding”, Virol. J., 3, 59 (2006).
23 Zhang, C., Liu, M.S., Xing, X.H., “Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase”, Appl. Microbiol. Biotechnol., 84 (3), 511-517 (2009).
24 Randers-Eichhorn, L., Albano, C.R., Sipior, J., Bentley, W.E., Rao, G., “On-line green fluorescent protein sensor with LED excitation”, Biotechnol. Bioeng., 55 (6), 921-926 (1997).
25 Chae, H.J., DeLisa, M.P., Cha, H.J., Li, J.C., Weigand, W.A., Valdes, J.J., Rao, G., Bentley, W.E., “On-line optimization and control of recombinant protein expression in high cell density Escherichia coli cultures using GFP-fusion monitoring”, Abstr. Pap. Am. Chem. S, 219, U213-U213 (2000).
26 Chae, H.J., DeLisa, M.P., Cha, H.J., Weigand, W.A., Rao, G., Bentley, W.E., “Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring”, Biotechnol. Bioeng., 69 (3), 275-285 (2000).
27 DeLisa, M.P., Li, J.C., Rao, G., Weigand, W.A., Bentley, W.E., “Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor”,Biotechnol. Bioeng., 65 (1), 54-64 (1999).
28 Zhang, C., Xing, X.H., “Quantification of a specific bacterial strain in an anaerobic mixed culture for biohydrogen production by the aerobic fluorescence recovery (AFR) technique”, Biochem. Eng. J., 39 (3), 581-585 (2008).
29 Kong, H.G., Choi, K.H., Heo, K.R., Lee, K.Y., Lee, H.J., Moon, B.J., Lee, S.W., “Generation of a constitutive green fluorescent protein expression construct to mark biocontrol bacteria using P43 promoter from Bacillus subtilis”, Plant Pathol. J., 25 (2), 136-141 (2009).
30 Li, Q., Chen, R., Li, W., Qiao, C.L., Wu, Y.J., “A genetically engineered Escherichia coli, expressing the fusion protein of green fluorescent protein and carboxylesterase B1, can be easily detected in the environment following degradation of pesticide residues”, Biotechnol. Lett., 29 (9), 1357-1362 (2007).
31 Skillman, L.C., Sutherland, I.W., Jones, M.V., Goulsbra, A., “Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms”, Microbiology, 144, 2095-2101 (1998).
32 Bizzarri, R., Serresi, M., Luin, S., Beltram, F., “Green fluorescent protein based pH indicators for in vivo use:A review”, Anal. Bioanal. Chem., 393 (4), 1107-1122 (2009).
33 Awaji, T., Hirasawa, A., Shirakawa, H., Tsujimoto, G., Miyazaki, S., “Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains”, Biochem. Biophys. Res. Commun., 289 (2), 457-462 (2001).
34 Takahashi, E., Takano, T., Nomura, Y., Okano, S., Nakajima, O., Sato, M., “In vivo oxygen imaging using green fluorescent protein”, Am. J. Physiol. Cell Physiol., 291 (4), C781-C787 (2006).
35 Garcia, J.R., Cha, H.J., Rao, G., Marten, M.R., Bentley, W.E., “Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors”, Microb. Cell Fact., 8, 6 (2009).
36 Lohman, J.R., Remington, S.J., “Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments”, Biochem., 47 (33), 8678-8688 (2008).
37 Hanson, G.T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R.A., Tsien, R.Y., Remington, S.J., “Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators”, J. Biol. Chem., 279 (13), 13044-13053 (2004).
38 Cannon, M.B., Remington, S.J., “Re-engineering redox-sensitive green fluorescent protein for improved response rate”, Protein Science, 15 (1), 45-57 (2006).
39 Heim, R., Cubitt, A.B., Tsien, R.Y., “Improved green fluorescence”, Nature, 373 (6516), 663-664 (1995).
40 Wurth, C., Guimard, N.K., Hecht, M.H., “Mutations that reduce aggregation of the Alzheimer’s A beta 42 peptide:an unbiased search for the sequence determinants of A beta amyloidogenesis”, J. Mol. Biol., 319 (5), 1279-1290 (2002).
41 Pedelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C., Waldo, G.S., “Engineering and characterization of a superfolder green fluorescent protein”, Nature Biotechnol., 24 (1), 79-88 (2006).
42 Cabantous, S., Terwilliger, T.C., Waldo, G.S., “Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein”, Nature Biotechnol., 23 (1), 102-107 (2005).
43 Reid, B.G., Flynn, G.C., “Chromophore formation in green fluorescent protein”, Biochem., 36 (22), 6786-6791 (1997).
44 Zhang, C., Xing, X.H., Lou, K., “Rapid detection of a gfp-marked Enterobacter aerogenes under anaerobic conditions by aerobic fluorescence recovery”, FEMS Microbiol. Lett., 249 (2), 211-218 (2005).
45 Lang, C., Schuler, D., “Expression of green fluorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria”, Appl. Environ. Microbiol., 74 (15), 4944-4953 (2008).
46 Tielker, D., Eichhof, I., Jaeger, K.E., Ernst, J.F., “Flavin mononucleotide-based fluorescent protein as an oxygen-independent reporter in Candida albicans and Saccharomyces cerevisiae”, Eukaryotic Cell, 8 (6), 913-915 (2009).
47 Bevis, B.J., Glick, B.S., “Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)”, Nature Biotechnol., 20 (1), 83-87 (2002).
48 French, S.A., Territo, P.R., Balaban, R.S., “Correction for inner filter effects in turbid samples:fluorescence assays of mitochondrial NADH”, Am. J. Physiol. Cell Physiol., 44 (3), C900-C909 (1998).
49 Kao, S.M., Asanov, A.N., Oldham, P.B., “A comparison of fluorescence inner-filter effects for different cell configurations”, Instrum. Sci. Technol., 26 (4), 375-387 (1998).
50 Konstantinov, K.B., Dhurjati, P., Vandyk, T., Majarian, W., Larossa, R., “Real-time compensation of the inner filter effect in high-density bioluminescent cultures”, Biotechnol. Bioeng., 42 (10), 1190-1198 (1993).
51 Srinivas, S.P., Mutharasan, R., “Inner filter effects and their interferences in the interpretation of culture fluorescence”, Biotechnol. Bioeng., 30 (6), 769-774 (1987).
52 Zanzotto, A., Boccazzi, P., Gorret, N., Van Dyk, T.K., Sinskey, A.J., Jensen, K.F., “In situ measurement of bioluminescence and fluorescence in an integrated microbioreactor”, Biotechnol. Bioeng., 93 (1), 40-47 (2006).
53 Zhang, C., Liu, M.S., Xing, X.H., “Correcting for the inner filter effect in measurements of fluorescent proteins in high-cell-density cultures”, Anal. Biochem., 390 (2), 197-202 (2009)
54 Su, W.W., Liu, B., Lu, W.B., Xu, N.S., Du, G.C., Tan, J.L., “Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures”, Biotechnol. Bioeng., 91 (2), 213-226 (2005).
55 Wang, G.J., Gentry, T.J., Grass, G., Josephson, K., Rensing, C., Pepper, I.L., “Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil”, FEMS Microbiol. Lett., 233 (2), 307-314 (2004).
56 Bohanec, B., Luthar, Z., Rudolf, K., “A protocol for quantitative analysis of green fluorescent protein-transformed plants, using multiparameter flow cytometry with cluster analysis”, Acta Biol. Cracov. Bot., 44, 145-153 (2002).
57 Valdivia, R.H., Ramakrishnan, L., “Applications of gene fusions to green fluorescent protein and flow cytometry to the study of bacterial gene expression in host cells”, Methods Enzymol., 326, 47-73 (2000).
58 Fujiki, Y., Tao, K., Bianchi, D.W., Giel-Moloney, M., Leiter, A.B., Johnson, K.L., “Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry:Comparison of transgenic strains and relevance for fetal cell microchimerism”, Cytometry, 73A (2), 111-118 (2008).
59 Jones, J.J., Bridges, A.M., Fosberry, A.P., Gardner, S., Lowers, R.R., Newby, R.R., James, P.J., Hall, R.M., Jenkins, O., “Potential of real-time measurement of GFP-fusion proteins”, Journal of Biotechnol., 109 (1/2), 201-211 (2004).
60 Miao, H., Ratnasingam, S., Pu, C.S., Desai, M.M., Sze, C.C., “Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context”, J. Microbiol. Methods, 76 (2), 111-119 (2009).
61 Ou, J., Furusawa, C., Yomo, T., Shimizu, H., “Analysis of stochasticity in promoter activation by using a dual-fluorescence reporter system”, Biosystems, 97 (3), 160-164 (2009).

[1]	Vesna Krsti?. Theoretical and experimental assessment of a novel method to establish the complete measurement range of the calorimeter and its limit of detection and quantification[J]. 中国化学工程学报, 2022, 44(4): 466-473.
[2]	Lingfeng Zhou, Junwu Wang, Wei Ge, Shiwen Liu, Jianhua Chen, Ji Xu, Limin Wang, Feiguo Chen, Ning Yang, Rongtao Zhou, Lin Zhang, Qi Chang, Philippe Ricoux, Alvaro Fernandez. Quantifying growth and breakage of agglomerates in fluid-particle flow using discrete particle method[J]. Chinese Journal of Chemical Engineering, 2018, 26(5): 914-921.
[3]	黄明涛, 陈韵妍, 刘建忠. Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀[J]. Chinese Journal of Chemical Engineering, 2014, 22(5): 559-569.
[4]	陈银, 邢新会, 叶逢春, 况莹. 重组大肠杆菌可溶表达并快速定量GFP-hepA融合蛋白[J]. , 2007, 15(1): 122-126.
[5]	周笑鹏, 史清洪, 邢新会, 孙彦. 快速纯化在大肠杆菌中表达的增强型绿色荧光蛋白 [J]. , 2006, 14(2): 229-234.