三片段高效GFP重组蛋白分裂系统的构建及功能研究

doi:10.3877/cma.j.issn.2095-7157.2025.04.009

绿色荧光蛋白(GFP)可被切割为不同片段并在空间接近时发生重组，因此被广泛用于研究蛋白相互作用。然而，针对天然产物与靶蛋白相互作用检测的高效GFP分裂体系仍然缺乏。本研究旨在构建并优化一种可用于检测天然产物–靶蛋白相互作用的三片段GFP分裂系统。

方法

本研究设计了一种三片段GFP分裂系统。采用雷帕霉素与FKBP12之间的高亲和力结合模型作为体系验证工具，在酵母细胞内评估该三片段GFP系统的重组效率与荧光信号。进一步通过对雷帕霉素进行氯代烷烃化学修饰以实现HaloTag标记，并对系统背景信号与荧光强度进行优化分析。

结果

实验结果表明，雷帕霉素的氯代烷烃修饰可显著增强三片段GFP系统的荧光信号。然而，该体系在细胞内仍存在较高的背景荧光，导致有效信号较弱，限制了其在高通量筛选中的应用。为此，本研究通过引入可溶性标签蛋白提高GFP1-9主片段的溶解性和结构稳定性，从而增强其与其他GFP片段的重组效率。此外，将HaloTag标记系统替换为亲和力更强的链霉亲和素-生物素体系后。

结论

本研究成功构建并系统优化了一种基于三片段GFP的天然产物–靶蛋白相互作用检测体系。通过提高GFP主片段溶解性并引入高亲和力标记系统，有效增强了信号强度并降低了背景干扰，为该体系在高通量筛选和天然产物功能研究中的应用奠定了基础。

关键词: 小分子标记系统, GFP重组蛋白, 天然产物

Objective

Green fluorescent protein (GFP) can be split into multiple fragments that reconstitute fluorescence upon spatial proximity, making it a powerful tool for probing biomolecular interactions.This study aimed to design and optimize a tripartite split-GFP system for monitoring natural product-target protein interactions in vivo.

Methods

The high-affinity interaction between rapamycin and FKBP12 was used as a model system to evaluate split-GFP reconstitution efficiency in yeast cells.A chloroalkane-modified rapamycin derivative was employed to enable HaloTag labeling, and fluorescence output was assessed.To improve system performance, solubility tags were introduced to enhance the stability of the GFP1-9 fragment, and the HaloTag labeling system was replaced with a streptavidin-biotin system with higher binding affinity.

Results

Chemical modification of rapamycin with a chloroalkane moiety significantly enhanced fluorescence intensity without compromising HaloTag labeling efficiency. Nevertheless, the tripartite split-GFP system exhibited substantial background fluorescence in vivo, resulting in weak effective signal and limiting its applicability in high-throughput screening. Improving the solubility and structural stability of the GFP1-9 core fragment increased reconstitution efficiency with the other GFP fragments. Furthermore, replacing the HaloTag system with the higher-affinity streptavidin-biotin labeling system led to a pronounced enhancement of fluorescence signal.

Conclusion

This study establishes and optimizes a tripartite split-GFP platform for detecting natural product-target protein interactions.By improving GFP fragment solubility and incorporating a high-affinity labeling system, signal intensity was significantly enhanced while background interference was reduced, supporting the potential application of this system in high-throughput screening and chemical biology research.GFP reconstitution has been well studied and applied widely as a report system.Since natural products are promising drug candidates, a robust split GFP report system can be valuable for drug development.

Key words: Small molecule report system, Split GFP system, Natural products

图1 三片段GFP分裂系统注：A：经烷基氯化物修饰的天然产物拟议结构，该修饰可通过Halo-Tag系统实现标记；B：三片段分裂GFP系统设计示意图。GFP10通过Halo-Tag与天然产物结合，GFP11则与靶蛋白融合。天然产物与靶蛋白的相互作用使GFP10和GFP11接近，从而与GFP1-9重构并产生绿色荧光信号，该信号可表示分子相互作用成功

图2 用于链霉亲和素–生物素系的分子合成和纯化注：A：用于合成生物素-雷帕霉素偶联物的酯化反应示意图。羧酸修饰的生物素与雷帕霉素上的羟基反应形成酯键；B:显示合成化合物纯化的HPLC色谱图。峰P3对应目标产物，其中生物素正确连接于预期位置。其他峰值为副产物，包括生物素连接位置错误或标记过量/不足的分子；C：链霉亲和素-GFP10融合蛋白变性纯化的SDS-PAGE分析。采用含6M尿素的裂解、洗涤及洗脱缓冲液进行纯化，证实成功分离出目标蛋白

图3 检测FKBP12-雷帕霉素相互作用的GFP分裂系统体外荧光信号注：测试了FKBP12-GFP11和GFP11-FKBP12两种构建体，以比较GFP11的N端和C端标签效果。阴性对照包括缺乏Halo标记的雷帕霉素的构建体，以及将FKBP12替换为MBP的构建体。阳性对照为直接GFP10-GFP11融合蛋白。荧光信号通过板式阅读器测定。FKBP12-雷帕霉素分裂GFP系统的信号在4 h后开始超过背景水平，并在24 h和48 h与阴性对照呈现明显分离，证实重组成功

图4 启动子强度对酵母内GFP分裂系统信号与背景比的影响注：FKBP12与GFP11融合蛋白分别在强启动子(质粒pKJ14)或弱启动子(质粒pKJ26)控制下表达。强启动子获得的最高信号背景比约为1.07，而弱启动子则产生更高比例，约1.26。GFP1-9与Halo标签GFP10均在标准强启动子(质粒pKJ17编码)下表达。阴性对照组仅含雷帕霉素而不含Halo标签系统。这些结果表明，在较弱启动子下表达FKBP12-GFP11可提高信号与背景的比值

图5 链霉亲和素-生物素基分裂GFP系统体外与体内评估注：A：链霉亲和素-生物素系分裂GFP系统与GFP10-GFP11融合蛋白阳性对照及两个缺乏Halo-雷帕霉素的阴性对照共同测试。荧光信号于23 h测定。链霉亲和素-生物素系统产生的荧光信号强度为Halo-Tag标签系统的2.36倍，且与阳性对照信号高度吻合；B：为确定最佳结合条件，采用GFP1-9(30 μM)、GFP10-链霉亲和素(5 μM)及FKBP12-GFP11(5 μM)进行生物素-雷帕霉素梯度滴定。于20 h测定荧光信号。经测定，生物素-雷帕霉素浓度达到5 μM时可获得最大信号输出；C：酵母体内链霉亲和素-生物素系统的测试显示，0 h与23 h时间点间荧光信号无显著变化，表明在该细胞条件下重组效率有限

图6 三片段GFP分裂系统的体内荧光信号比较注：A：质粒pKJ21-94表达额外GFP1-9基因后，观察到荧光信号相较于正常GFP1-9表达的质粒pKJ21有显著增强。此外我们展示了在不同OD条件下的pKJ21-94和pKJ21的比较(b)在融合MBP和GFP1-9后，GFP分裂系统的信号与背景比值通过与阴性对照组(20 μM雷帕霉素处理且无Halo标签)进行比较计算得出：25 h时，原始分裂GFP系统(pKJ17-26)比值为1.26，而MBP融合变体(pKJ17-109)比值为1.19，表明未显著改善

[1]	Atanasov AG，Zotchev SB，Dirsch VM，et al.Natural Products in Drug Discovery：Advances and Opportunities[J]．Nat Rev Drug Discov，2021，20(3)：200-216.
[2]	Vane JR.Inhibition of Prostaglandin Synthesis as a Mechanism of Action for Aspirin-like Drugs[J]．Nature New Biol，1971，231 (25)：232- 235.
[3]	Vane JR，Botting RM.The Mechanism of Action of Aspirin[J]．Thromb Res，2003，110(5-6)：255-258.
[4]	Jordan MA，Wilson L．Microtubules as a Target for Anticancer Drugs [J]．Nat Rev Cancer，2004, 4(4)：253-265.
[5]	Tsien RY.The Green Fluorescent Protenin[J]．Annu Rev Biochem, 1998，67(1)：509-544.
[6]	Paulmurugan R，Gambhir SS.Monitoring Protein-Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation[J]．Anal Chem，2003，75(7)：1584-1589.
[7]	Lukyanov KA.Fluorescent proteins for a brighter science[J]．Biochem Biophys Res Commun，2022，10(633)：29-32.
[8]	Park Ki Sung，Cha Hanvit，Niu Jia，et al.DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels[J]．Nucleic Acids Res，2024，52(14)：8063-8071.
[9]	Chen J，Zheng XF，Brown EJ，et al.Identification of an 11-kDa FKBP12- Rapamycin-Binding Domain within the 289-kDa FKBP12- Rapamycin-Associated Protein and Characterization of a Critical Serine Residue[J]．Proc Natl Acad Sci，1995，92(11)：4947-4951.
[10]	Brown EJ，Albers MW，Bum Shin T，et al.A Mammalian Protein Targeted by G1-Arresting Rapamycin-Receptor Complex[J]．Nature，1994, 369(6483)：756-758.
[11]	Cabantous S，Nguyen HB，Pedelacq JD，et al.A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association[J]．Sci Rep, 2013，3(1)：2854.
[12]	Kapust RB，Waugh DS.Escherichia Coli Maltose-binding Protein Is Uncommonly Effective at Promoting the Solubility of Polypeptides to Which It Is Fused[J]．Protein Sci，1999，8(8)：1668-1674.
[13]	Erdmann RS，Baguley SW，Richens JH，et al.Labeling Strategies Matter for Super-Resolution Microscopy：A Comparison between HaloTags and SNAP-Tags[J]．Cell Chem Biol，2019，26(4)：584-592.e6.

[1]	李懿雪, 潘婷, 林焕彩, 周燕. 植物源性天然产物在口腔细菌感染性疾病中的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2023, 17(03): 188-196.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Split-GFP-based three fragment recombination protein system designation and functional study

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Split-GFP-based three fragment recombination protein system designation and functional study