Construction and Functional Investigation of Fusion Expression Vectors Harboring StayGold, a Novel Fluorescent Protein Encoding Gene, and Antibiotic Resistance Genes

DOU Yuhan; GU Feng

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Journal of Hunan Normal University(Medical Science) ›› 2026, Vol. 23 ›› Issue (1) : 1-7.

Construction and Functional Investigation of Fusion Expression Vectors Harboring StayGold, a Novel Fluorescent Protein Encoding Gene, and Antibiotic Resistance Genes

DOU Yuhan, GU Feng

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Abstract

Objective To construct vectors that express StayGold green fluorescent protein in both prokaryotic and eukaryotic cells, while simultaneously expressing functional fused antibiotic resistance genes. Method The StayGold coding sequence was inserted at different positions within the antibiotic resistance gene to construct corresponding vectors. The prokaryotic vectors were transformed into E. coli DH10B, while the eukaryotic vectors were transfected into HEK293T cells. Antibiotic selection was used to investigate the expression of the antibiotic resistance gene. Fluorescence microscopy was harnessed to observe the samples and detect the expression of StayGold, with its fluorescence intensity compared to that of EGFP. After storing the bacterial culture samples in a refrigerator at 4℃ for one month, the fluorescence intensity was re-measured to compare the low-temperature stability of the fusion proteins StayGold and EGFP. Results In all prokaryotic vectors, StayGold was expressed normally. Only when StayGold was inserted into the C-terminus of the AmpR gene with or without a peptide linker (32 residues, 32AA), the transformed E. coli DH10B exhibited resistance to ampicillin. Moreover, its fluorescence intensity was comparable to that of the control group EGFP. However, after one month, the fluorescence intensity of the experimental group StayGold was higher than that of EGFP. In all eukaryotic vectors, StayGold was also expressed normally. When StayGold was inserted into the C-terminus of the PuroR or HygroR sequence, and a 32AA linker was added, the transfected HEK293T cells showed tolerance to the corresponding antibiotics. Conclusion Inserting StayGold into the C-terminus of the antibiotic resistance gene encoding protein and adding a linker can achieve functional fusion of fluorescence and resistance in both prokaryotic and eukaryotic cells. In E. coli, StayGold exhibits stronger low-temperature stability compared to EGFP.

Key words

StayGold / prokaryotic expression vector / eukaryotic expression vector / fusion protein

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DOU Yuhan, GU Feng. Construction and Functional Investigation of Fusion Expression Vectors Harboring StayGold, a Novel Fluorescent Protein Encoding Gene, and Antibiotic Resistance Genes[J]. Journal of Hunan Normal University(Medical Science). 2026, 23(1): 1-7

References

[1] SHIMOMURA O, JOHNSON F H, SAIGA Y.Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea[J]. J Cell Comp Physiol, 1962, 59: 223-239.
[2] CHALFIE M, TU Y, EUSKIRCHEN G, et al.Green fluorescent protein as a marker for gene expression[J]. Science (New York, NY), 1994, 263(5148): 802-805.
[3] MURAKAMI M, MURAKAMI A M, ITAGAKI S.A dual prokaryotic (E. coli) expression system (pdMAX)[J]. PloS One, 2021, 16(10): e0258553.
[4] BLUMENSTOCK S, SCHULZ-TRIEGLAFF E K, VOELKL K, et al. Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease[J]. EMBO J, 2021, 40(19): e107260.
[5] BAI J, KOOS D S, STEPANIAN K, et al. Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids[J]. Dis Model Mech, 2023, 16(11): dmm050193.
[6] BETZIG E, PATTERSON G H, SOUGRAT R, et al.Imaging intracellular fluorescent proteins at nanometer resolution[J]. Science (New York, NY), 2006, 313(5793): 1642-1645.
[7] OUYANG S, QIN W M, NIU Y J, et al.An EGFP Knock-in Zebrafish Experimental Model Used in Evaluation of the Amantadine Drug Safety During Early Cardiogenesis[J]. Front Cardiovasc Med, 2022, 9: 839166.
[8] KUNTE M M, CHOUDHURY S, MANHEIM J F, et al.ER stress is involved in T17M rhodopsin-induced retinal degeneration[J]. Invest Ophthalmol Vis Sci, 2012, 53(7): 3792-3800.
[9] HIRANO M, ANDO R, SHIMOZONO S, et al.A highly photostable and bright green fluorescent protein[J]. Nature biotechnol, 2022, 40(7): 1132-1142.
[10] ANDO R, SHIMOZONO S, AGO H, et al.StayGold variants for molecular fusion and membrane-targeting applications[J]. Nat methods, 2024, 21(4): 648-656.
[11] Goedhart J, Gadella T W J Jr. Breaking up the StayGold dimer yields three photostable monomers[J]. Nat methods, 2024, 21(4): 558-559.
[12] LIN Y, EXELL J, LIN H, et al.Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions[J]. J Phys Chem B, 2024, 128(23): 5590-5600.
[13] FRANZKOCH R, WILKENING S, LISS V, et al.Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold[J]. Heliyon, 2024, 10(7): e28055.
[14] HIRANO M, YONEMARU Y, SHIMOZONO S, et al.StayGold photostability under different illumination modes[J]. Scie Rep, 2024, 14(1): 5541.
[15] VIOLA G, JACOBS K A, LEMIÈRE J, et al. Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells[J]. bioRxiv[Preprint]2024. doi: 10.1101/2024.09.16.613379.
[16] SAKAMOTO K, YAMAMOTO Y, INABA H, et al.Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide[J]. ACS synthet biol, 2024, 13(6): 1842-1850.
[17] GANINI D, LEINISCH F, KUMAR A, et al.Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells[J]. Redox biol, 2017, 12: 462-468.
[18] IVORRA-MOLLA E, AKHULI D, MCANDREW M B L, et al. A monomeric StayGold fluorescent protein[J]. Nat Biotechnol, 2024, 42(9): 1368-1371.
[19] ZHANG H, LESNOV G D, SUBACH O M, et al.Bright and stable monomeric green fluorescent protein derived from StayGold[J]. Nat methods, 2024, 21(4): 657-665.
[20] TIAN R, REHM F B H, CZERNECKI D, et al. Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli[J]. Science (New York, NY), 2024, 383(6681): 421-426.