WO2024087539A1 - 一种增强型StayGold黄色荧光蛋白及其应用 - Google Patents

一种增强型StayGold黄色荧光蛋白及其应用 Download PDF

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WO2024087539A1
WO2024087539A1 PCT/CN2023/089029 CN2023089029W WO2024087539A1 WO 2024087539 A1 WO2024087539 A1 WO 2024087539A1 CN 2023089029 W CN2023089029 W CN 2023089029W WO 2024087539 A1 WO2024087539 A1 WO 2024087539A1
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staygold
fluorescent protein
yellow fluorescent
enhanced
protein
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PCT/CN2023/089029
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French (fr)
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王峰
杨益虎
陈倩
桂文君
吕志佳
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无锡佰翱得生物科学股份有限公司
佰翱得(无锡)新药开发有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02B20/00—Energy efficient lighting technologies, e.g. halogen lamps or gas discharge lamps

Definitions

  • the present disclosure relates to the field of recombinant protein technology, and in particular to an enhanced StayGold yellow fluorescent protein and applications thereof.
  • the purpose of the present disclosure is to overcome some application limitations of the existing StayGold green fluorescent protein and to provide an enhanced StayGold yellow fluorescent protein and its application.
  • the present disclosure provides an enhanced StayGold yellow fluorescent protein, named YStayGold.
  • the sequence of the enhanced StayGold yellow fluorescent protein is a mutant sequence of a wild-type StayGold protein, and the mutation in the mutant sequence is K192Y, that is, the 192nd amino acid residue of the wild-type StayGold protein is mutated from lysine to tyrosine, so that the green fluorescent StayGold protein can be converted into yellow YStayGold.
  • amino acid sequence of the enhanced StayGold yellow fluorescent protein is shown as SEQ ID NO. 1. In other embodiments, the amino acid sequence of the enhanced StayGold yellow fluorescent protein is shown as SEQ ID NO. 2.
  • the present disclosure also provides a polynucleotide encoding the enhanced StayGold yellow fluorescent protein.
  • sequence of the polynucleotide is as shown in SEQ ID NO.3.
  • the present disclosure also provides a recombinant plasmid, which is an expression vector containing the polynucleotide and capable of translating and expressing the enhanced StayGold yellow fluorescent protein.
  • the expression vector is a pET28a vector.
  • the present disclosure also provides a protein expression system, which is an Escherichia coli BL21 strain into which the above-mentioned recombinant plasmid is transferred.
  • the present disclosure also provides a method for preparing the above-mentioned enhanced StayGold yellow fluorescent protein, constructing the pET28a-6His-Strep II-TEV-GG-YStayGold (K192Y) recombinant plasmid and transforming BL21 (DE3) Escherichia coli competent cells to express the recombinant plasmid, and then using affinity chromatography His FF enrichment and purification to obtain the yellow fluorescent protein.
  • the present disclosure also provides an application of the enhanced StayGold yellow fluorescent protein in detecting protein labeling and expression localization.
  • the beneficial effects of the present disclosure are as follows: Based on the existing research results, the present disclosure expands the application of StayGold protein and develops a new yellow YStayGold protein based on green fluorescent protein.
  • the yellow fluorescent protein has strong thermal stability, with a Tm of about 95°C, which is 15°C higher than the Tm value of YFP commonly used on the market, and the fluorescence intensity is also 8 times higher.
  • the YStayGold yellow fluorescent protein has a broader application market than the YFP yellow fluorescent protein commonly used on the market.
  • Figure 1 is a small amount expression detection diagram of YStayGold
  • Figure 2 is a color comparison chart of YStayGold, StayGold and YFP;
  • Figure 3 is a diagram of YStayGold nickel column purification
  • FIG4 is a diagram of YFP nickel column purification
  • FIG5 is a graph showing the determination of excitation wavelength and emission wavelength of YStayGold, StayGold and YFP;
  • FIG6 is a diagram showing the detection of Tm values of YStayGold and YFP yellow fluorescent protein
  • FIG. 7 is a fluorescence intensity detection diagram of YStayGold and YFP yellow fluorescent protein.
  • the present invention uses gene synthesis technology to obtain the StayGold gene, which is synthesized on the pET28a vector (Ubao Bio, catalog number: VT1207) to obtain the pET28a-6His-Strep II-TEV-GG-StayGold plasmid.
  • the YStayGold plasmid uses the StayGold gene as a template to design the K192Y mutant amino acid primer:
  • Reverse primer TTTGTGTGTATTGATAACGAATCCAGTGAT (SEQ ID NO.7);
  • the pET28a-6His-Strep II-TEV-GG-YStayGold(K192Y) recombinant plasmid was obtained. All recombinant plasmids were sequenced correctly.
  • the amino acid sequence of YStayGold is shown in SEQ ID NO.1, and the nucleotide sequence encoding the YStayGold protein is shown in SEQ ID NO.3.
  • the recombinant plasmid pET28a-6His-Strep II-TEV-GG-YStayGold (K192Y) constructed successfully and sequenced correctly was transformed into BL21 (DE3) Escherichia coli competent cells (Weidi Biotechnology, Cat. No.: EC1002), and a single clone plaque was picked to 5mL LB liquid culture medium, cultured at 37°C, and a small amount of bacterial solution was fixed with loading buffer when the bacterial solution OD 600 reached 0.6-0.8, and a small amount of bacterial solution was added to glycerol and frozen to -80°C.
  • the strain that clearly expressed YStayGold was inoculated into 50 mL LB liquid medium and cultured at 37°C overnight.
  • the bacteria cultured overnight were inoculated into 1 L LB liquid medium at a ratio of 1:100 and cultured at 37°C until the OD600 of the bacterial solution was 0.6-0.8.
  • 0.5 mM IPTG was added and cultured at 15°C overnight, and the bacteria were collected by centrifugation at 5000 rpm.
  • the collected bacterial blocks were weighed, and the corresponding volume of lysis buffer (50mM Tris-HCl (pH 7.5), 150mM NaCl) was added at a ratio of 1:10.
  • the bacteria were broken using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000rpm.
  • the protein was enriched and purified using affinity chromatography His FF. Before purification, the His FF column was balanced with lysis buffer. After all the cell supernatants were hung on the column, they were eluted with different gradients of imidazole solution, and the proteins eluted with different gradients of imidazole were collected for SDS-PAGE detection. The purification results are shown in Figure 3. The YStayGold protein was clearly expressed and had good purity.
  • YFP recombinant protein was expressed in the prokaryotic expression system (protein sequence see SEQ ID NO.4).
  • the expression method of YFP protein is the same as that of YStayGold. Because the N-terminus of YFP protein carries a Flag tag, it is purified using an Anti-Flag G1 column.
  • the specific scheme is as follows: the collected bacterial blocks are weighed, and the corresponding volume of lysis buffer (100mM Hepes (pH 7.5), 150mM NaCl) is added at a ratio of 1:10.
  • the bacteria are broken by a high-pressure homogenizer, and the supernatant is collected by high-speed centrifugation at 16000rpm.
  • the protein is enriched and purified using affinity chromatography Anti-Flag G1. Before purification, the Anti-Flag G1 column is balanced with lysis buffer. After all the cell supernatants are hung on the column, 200ng/ul of polypeptide is used for elution. The eluted protein is collected for SDS-PAGE detection.
  • the purification results are shown in Figure 4.
  • the YFP protein is clearly expressed and has good purity. The colors are shown in Figure 2. In terms of color, YFP is comparable to YStayGold.
  • YStayGold According to the color of the purified protein, YStayGold can be roughly judged as a yellow fluorescent protein.
  • a microplate reader was used for determination.
  • the excitation and emission wavelengths of the YFP protein were also determined. The specific operation process is as follows:
  • the concentration of StayGold, YstayGold and YFP proteins to be tested was diluted to 0.50 mg/ml, and the maximum absorption wavelengths of the two proteins were scanned respectively with a full wavelength scanner.
  • the experimental results are shown in Figure 5, where the maximum absorption wavelength of StayGold is 480 nm, the maximum absorption wavelength of YStayGold is 500 nm, and the maximum absorption wavelength of YFP is 510 nm. After determining the absorption wavelengths of the three proteins, the corresponding wavelengths were used as excitation light to detect the maximum emission wavelengths of the three proteins.
  • the results in Figure 5 show that the maximum emission wavelengths of StayGold, YStayGold and YFP are 510nm, 515nm and 521nm, respectively.
  • the excitation wavelength of StayGold green fluorescent protein is 480nm and the emission wavelength is 510nm; the excitation wavelength of YStayGold protein is 500nm and the emission wavelength is 515nm.
  • the excitation wavelength of YFP is 510nm and the emission wavelength is 521nm. From the excitation wavelength and emission wavelength of the two proteins StayGold and YStayGold, the wavelength of YStayGold is higher than that of StayGold protein, which also shows that the mutation of lysine at position 192 to tyrosine causes the green fluorescence of StayGold to turn into yellow fluorescence.
  • the purified YStayGold protein was selected and compared with the fluorescence intensity of the purified YFP protein.
  • MGSS is the sequence on the pET28a vector
  • HHHHHH is the 6 ⁇ His tag
  • WSHPQFEK is the Strep II tag
  • ENLYFQG is the TEV protease cleavage site
  • GG is the linker.
  • SEQ ID NO.2 Enhanced StayGold yellow fluorescent protein amino acid sequence (artificial sequence)
  • SEQ ID NO.4 (Jellyfish, Cytaeis uchidae)
  • SEQ ID NO.5 Amino acid sequence of wild-type StayGold protein (jellyfish, Cytaeis uchidae)

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Abstract

一种增强型StayGold黄色荧光蛋白及其应用，涉及重组蛋白技术领域。该增强型StayGold黄色荧光蛋白为StayGold的氨基酸序列中包含了192位赖氨酸突变酪氨酸氨基酸，即可将绿色的荧光StayGold蛋白变成黄色的YStayGold。提供的YStayGold的最大激发波长为500nm，最大发射波长为515nm，且具有很强的热稳定性，Tm大于95℃。比市场上常用的YFP黄色荧光蛋白的Tm值高出15℃，且荧光强度也高出8倍，说明YStayGold黄色荧光蛋白明显比市场上常用的YFP黄色荧光蛋白具有更广阔的应用市场。

Description

一种增强型StayGold黄色荧光蛋白及其应用
优先权和相关申请
本公开要求2022年10月28日提交的名称为“一种增强型StayGold黄色荧光蛋白及其应用”的中国专利申请202211332706.9的优先权，该申请包括附录在内的全部内容作为参考并入本公开。
技术领域
本公开涉及重组蛋白技术领域，具体涉及一种增强型StayGold黄色荧光蛋白及其应用。
背景技术
运用荧光蛋白监测细胞活动、标记蛋白表达的技术已经深入蛋白质组学的研究。最早的荧光蛋白是在1962年由下村修等人在Aequorea victoria水母中发现的绿色荧光蛋白。在绿色荧光蛋白广泛应用后，科学人员发现单纯的绿色荧光蛋白通常不能完全满足研究需要，比如在很多实验中需要同时标记两种以上的细胞或蛋白。因此，科研人员后期通过研究绿色荧光蛋白的结构特点，通过对绿色荧光蛋白的某些氨基酸进行突变，从而获得了不同颜色的变种蛋白，如黄色、蓝色、青色以及红色等。多种颜色荧光蛋白的发现不仅为科研提供了方便，同时也扩展了荧光蛋白的应用市场。
Hirano，Masahiko等人在2022年从Cytaeis uchidae水母中发现了一种新的绿色荧光蛋白StayGold，该荧光蛋白的光稳定性比目前常用的荧光蛋白都高出一个数量级，已经很好地应用于观察内质网的动态成像[Hirano，Masahiko et al.“A highly photostable and bright green fluorescent protein.”Nature biotechnology，10.1038/s41587-022-01278-2.25Apr.2022]，该荧光蛋白比目前常用的绿色荧光蛋白具有更强的光稳定性，提示着其具有更广泛的应用市场。目前对于该绿色荧光蛋白的开发研究还欠缺，因其绿色荧光稳定性比其他荧光蛋白要强，因此基于其绿色荧光设计出其变种蛋白，也具有很高的市场应用价值。
发明内容
本公开的目的在于克服现有StayGold绿色荧光蛋白的一些应用限制，提供了一种增强型StayGold黄色荧光蛋白及其应用。
本公开通过以下技术方案来实现上述目的：
本公开提供一种增强型StayGold黄色荧光蛋白，命名为YStayGold，该增强型StayGold黄色荧光蛋白序列为野生型StayGold蛋白的突变序列，且所述突变序列中的突变为K192Y，即野生型StayGold蛋白的第192位氨基酸残基由赖氨酸突变成酪氨酸，即可将绿色的荧光StayGold蛋白变成黄色的YStayGold。
在一些实施方式中，所述增强型StayGold黄色荧光蛋白的氨基酸序列如SEQ ID NO.1所示。在另一些实施方式中，所述增强型StayGold黄色荧光蛋白的氨基酸序列如SEQ ID NO.2所示。
本公开还提供一种多核苷酸，所述多核苷酸编码上述增强型StayGold黄色荧光蛋白。
在一些实施方式中，所述多核苷酸的序列如SEQ ID NO.3所示。
本公开还提供一种重组质粒，所述重组质粒为含有上述多核苷酸且能够翻译表达出上述增强型StayGold黄色荧光蛋白的表达载体。
在一些实施方式中，所述表达载体为pET28a载体。
本公开还提供一种蛋白表达系统，为转入上述重组质粒的大肠杆菌BL21菌株。
本公开还提供一种上述增强型StayGold黄色荧光蛋白的制备方法，构建pET28a-6His-Strep II-TEV-GG-YStayGold(K192Y)重组质粒并转化BL21(DE3)大肠杆菌感受态细胞中表达所述重组质粒，再使用亲和层析His FF富集纯化获得所述黄色荧光蛋白。
本公开还提供一种上述增强型StayGold黄色荧光蛋白在检测蛋白的标记、表达定位中的应用。
本公开的有益效果在于：本公开根据现有的研究结果，扩展了StayGold蛋白的应用，在绿色荧光蛋白的基础上开发了一种新的黄色YStayGold蛋白。该黄色荧光蛋白有很强的热稳定性，Tm约为95℃，比市场上常用的YFP的Tm值高出15℃，且荧光强度也高8倍。YStayGold黄色荧光蛋白比市场上常用的YFP黄色荧光蛋白具有更广阔的应用市场。
附图说明
图1为YStayGold小量表达检测图；
图2为YStayGold与StayGold、YFP颜色对比图；
图3为YStayGold镍柱纯化图；
图4为YFP镍柱纯化图；
图5为YStayGold与StayGold、YFP激发波长和发射波长测定图；
图6为YStayGold与YFP黄色荧光蛋白Tm值检测图；
图7为YStayGold与YFP黄色荧光蛋白荧光强度检测图。
具体实施方式
下面结合附图对本申请作进一步详细描述，有必要在此指出的是，以下具体实施方式只用于对本申请进行进一步的说明，不能理解为对本申请保护范围的限制，该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。
1、材料
本实施例所用方法如无特别说明均为本领域的技术人员所知晓的常规方法，所用的试剂等材料，如无特别说明，均为市售购买产品。
2、方法
2.1 YStayGold蛋白质粒构建及表达
2.1.1 YStayGold质粒构建
本公开用基因合成技术获得StayGold的基因，其基因是合成在pET28a载体(优宝生物，货号：VT1207)上，获得pET28a-6His-Strep II-TEV-GG-StayGold质粒。YStayGold的质粒是以StayGold的基因为模板，设计K192Y突变氨基酸引物：
正向引物：ATCACTGGATTCGTTATCAATACACACAAA(SEQ ID NO.6)；
反向引物：TTTGTGTGTATTGATAACGAATCCAGTGAT(SEQ ID NO.7)；
按照常规定点突变PCR的方法，获得pET28a-6His-Strep II-TEV-GG-YStayGold(K192Y)重组质粒。所有重组质粒均测序正确。YStayGold的氨基酸序列如SEQ ID NO.1所示，编码YStayGold蛋白的核苷酸序列如SEQ ID NO.3所示。
2.1.2YStayGold小量表达
将构建成功且测序正确的pET28a-6His-Strep II-TEV-GG-YStayGold(K192Y)重组质粒转化BL21(DE3)大肠杆菌感受态细胞(唯地生物,货号：EC1002)，挑取单克隆菌斑至5mL LB液体培养基中，37℃培养，待菌液OD600至0.6-0.8时取少量菌液用loading buffer进行固定，并取少量菌液加入甘油冻至-80℃，剩余菌液加入0.5mM IPTG诱导4个小时后，收集菌体并取诱导后菌液进行SDS-PAGE检测。根据SDS-PAGE结果可知YStayGold有明显表达(图1)。
2.2 YStayGold蛋白质纯化
2.2.1 YstayGold大量表达
将明显表达YStayGold的菌株接种至50mL LB液体培养基中37℃培养过夜，将过夜培养的细菌按1:100的比例接至1L LB液体培养基中，37℃培养至菌液OD600为0.6-0.8时加入0.5mM IPTG 15℃培养过夜，5000rpm离心收集菌体。
2.2.2 YStayGold重组蛋白纯化
将收集的菌块进行称重，按照1:10比例加入相应体积的裂解缓冲液(50mM Tris-HCl(pH 7.5)，150mM NaCl)，使用高压均质机破碎菌体，16000rpm高速离心收集上清。使用亲和层析His FF富集纯化蛋白，纯化前先用裂解buffer平衡His FF柱，将所有细胞上清挂柱后，用不同梯度的咪唑溶液洗脱，收集不同梯度咪唑洗脱下的蛋白进行SDS-PAGE检测，纯化结果如图3所示，YStayGold蛋白明显表达、纯度较好。
将YStayGold与StayGold蛋白的颜色进行对比，见图2，根据图2的结果可知StayGold蛋白颜色为绿色，YstayGold蛋白颜色为黄色，表明192位的赖氨酸突变成酪氨酸后，获得了绿色StayGold的变种黄色荧光蛋白YStayGold。
2.2.3 YFP重组蛋白纯化
为了将YStayGold与其他黄色荧光蛋白的性能进行比较，同样地在原核表达系统中表达了YFP重组蛋白(蛋白序列见SEQ ID NO.4)。YFP蛋白的表达方法与YStayGold相同，因为YFP蛋白N端带有Flag标签，因此纯化时是用Anti-Flag G1柱子进行纯化，具体方案为：将收集的菌块进行称重，按照1:10比例加入相应体积的裂解缓冲液(100mM Hepes(pH 7.5)，150mM NaCl)，使用高压均质机破碎菌体，16000rpm高速离心收集上清。使用亲和层析Anti-Flag G1富集纯化蛋白，纯化前先用裂解buffer平衡Anti-Flag G1柱，将所有细胞上清挂柱后，用200ng/ul的多肽洗脱，收集洗脱下的蛋白进行SDS-PAGE检测，纯化结果如图4所示，YFP蛋白明显表达、纯度较好。其颜色见图2，从颜色上看，YFP与YStayGold颜色相当。
2.3 YStayGold全波长扫描
根据纯化后蛋白的颜色可以大致判断YStayGold为黄色荧光蛋白，为了进一步测定YStayGold的激发和发射波长，用酶标仪进行了测定。同时也对YFP蛋白的激发波长和发射波长进行了测定，具体操作流程如下：
将待检测的StayGold、YstayGold和YFP蛋白浓度稀释为0.50mg/ml，用全波长扫描仪分别扫描两个蛋白的最大吸收波长，实验结果见图5，其中StayGold的最大吸收波长为480nm、YStayGold的最大吸收波长为500nm、YFP的最大吸收波长为510nm。在 确定好三个蛋白的吸收波长后，分别用相应的波长作为激发光，检测三个蛋白的最大发射波长，图5结果显示StayGold的最大发射波长为510nm、YStayGold的最大发射波长为515nm、YFP的最大发射波长为521nm。因此StayGold绿色荧光蛋白的激发波长为480nm，发射波长为510nm；YStayGold蛋白的激发波长为500nm，发射波长为515nm。YFP的激发波长为510nm，发射波长为521nm。从StayGold和YStayGold两个蛋白的激发波长和发射波长来看，YStayGold的波长比StayGold蛋白要高，也能说明192位的赖氨酸突变成酪氨酸后，使StayGold绿色荧光转变成黄色荧光。
2.4 YStayGold热稳定检测
为了研究YStayGold蛋白的热稳定性，将其与市场上常用的黄色荧光蛋白YFP进行Tm值的比较。具体的实验操作如下：
取20μL蛋白浓度稀释为0.50mg/mL的上述2种蛋白分别加到384孔实验板中，震荡离心后，将实验板置于取样架上，使用Nano DSF毛细管吸样，保证样品充满整个毛细管。将毛细管放入nanoDSF仪器中，设置初始温度为20℃，以每分钟升温2.0℃的速度最终上升到90℃终止。仪器将会依照设置好的参数进行升温和实时检测。Tm值测试的结果如图6所示，YFP的Tm值为79.1℃；YStayGold的Tm值太高，超出了仪器检测的最高值，根据其Tm的曲线来看，其Tm值约为95℃。实验表明本公开的YStayGold的热稳定性比市场上常用的黄色荧光蛋白要好，具有更好的应用场景。
2.5 YStayGold蛋白荧光强度检测
为了研究YStayGold的荧光强度，选择纯化后的YStayGold蛋白，同时与纯化后的YFP蛋白进行荧光强度的比较。
具体的实验操作如下：
分别取10μL 2种不同的荧光蛋白于384孔的反应板中，加入10μL反应缓冲液(50mM Tris-HCl pH 8.0，150mM NaCl)，用全波长扫描仪进行读数，YStayGold设置激发波长为500nm，发射波长为510nm、YFP设置激发波长为510nm，发射波长为521nm。实验数据如图7所示，YStayGold的荧光信号比YFP高出8倍。表明本公开的YStayGold的荧光强度比市场上常用的黄色荧光蛋白要强，具有更广泛的应用场景。
SEQ ID NO.1(人工序列)
其中，MGSS为pET28a载体上序列，HHHHHH为6×His标签，WSHPQFEK为Strep II标签，ENLYFQG为TEV蛋白酶切割位点，GG为接头。
SEQ ID NO.2：增强型StayGold黄色荧光蛋白氨基酸序列(人工序列)
SEQ ID NO.3(人工序列)
SEQ ID NO.4(水母，Cytaeis uchidae)
SEQ ID NO.5：野生型StayGold蛋白氨基酸序列(水母，Cytaeis uchidae)
以上所述实施例仅表达了本公开的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本公开专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本公开构思的前提下，还可以做出若干变形和改进，这些都属于本公开的保护范围。

Claims (9)

  1. 一种增强型StayGold黄色荧光蛋白，其特征在于，该增强型StayGold黄色荧光蛋白序列为野生型StayGold蛋白的突变序列，且所述突变序列中的突变为K192Y，即野生型StayGold蛋白的第192位氨基酸残基由赖氨酸突变成酪氨酸。
  2. 根据权利要求1所述的增强型StayGold黄色荧光蛋白，其特征在于，所述增强型StayGold黄色荧光蛋白的氨基酸序列如SEQ ID NO.1所示；和/或，
    所述增强型StayGold黄色荧光蛋白的氨基酸序列如SEQ ID NO.2所示；
    优选地，所述增强型StayGold黄色荧光蛋白的氨基酸序列如SEQ ID NO.1所示。
  3. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1-2任一项所述的增强型StayGold黄色荧光蛋白。
  4. 根据权利要求3所述的一种多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO.3所示。
  5. 一种重组质粒，其特征在于，所述重组质粒为含有如权利要求3-4任一项所述的多核苷酸且能够翻译表达出如权利要求1-2任一项所述增强型StayGold黄色荧光蛋白的表达载体。
  6. 根据权利要求5所述的一种重组质粒，其特征在于，所述表达载体为pET28a载体。
  7. 一种蛋白表达系统，其特征在于，为转入权利要求6所述重组质粒的大肠杆菌BL21菌株。
  8. 一种如权利要求1-2任一项所述的增强型StayGold黄色荧光蛋白的制备方法，其特征在于，构建pET28a-6His-Strep II-TEV-GG-黄色荧光蛋白的重组质粒并转化BL21(DE3)大肠杆菌感受态细胞中表达所述重组质粒，再使用亲和层析His FF富集纯化获得所述黄色荧光蛋白。
  9. 一种如权利要求1-2任一项所述的增强型StayGold黄色荧光蛋白在检测蛋白的标记、表达定位中的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060167225A1 (en) * 2002-01-22 2006-07-27 Gurskaya Nadejda G Novel fluorescent protein from aequorea coerulscens and methods for using the same
CN105585625A (zh) * 2014-10-31 2016-05-18 北京义翘神州生物技术有限公司 一种增强型绿色荧光蛋白
CN108834418A (zh) * 2016-03-10 2018-11-16 国立大学法人大阪大学 荧光蛋白质
CN110964088A (zh) * 2018-09-30 2020-04-07 中国科学院生物物理研究所 一种可基因编码的人工光合作用蛋白质及其应用
WO2022215532A1 (ja) * 2021-04-07 2022-10-13 国立研究開発法人理化学研究所 蛍光特性を示す新規なポリペプチド、およびその利用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110330558B (zh) * 2019-08-05 2020-09-22 博迈德生物科技（固安）有限公司 一种黄色荧光蛋白及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060167225A1 (en) * 2002-01-22 2006-07-27 Gurskaya Nadejda G Novel fluorescent protein from aequorea coerulscens and methods for using the same
CN105585625A (zh) * 2014-10-31 2016-05-18 北京义翘神州生物技术有限公司 一种增强型绿色荧光蛋白
CN108834418A (zh) * 2016-03-10 2018-11-16 国立大学法人大阪大学 荧光蛋白质
CN110964088A (zh) * 2018-09-30 2020-04-07 中国科学院生物物理研究所 一种可基因编码的人工光合作用蛋白质及其应用
WO2022215532A1 (ja) * 2021-04-07 2022-10-13 国立研究開発法人理化学研究所 蛍光特性を示す新規なポリペプチド、およびその利用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 18 June 2022 (2022-06-18), ANONYMOUS: "fluorescent protein [synthetic construct", XP093164598, retrieved from NCBI Database accession no. BCN13401.1 *
HIRANO, M. ET AL.: "A highly photostable and bright green fluorescent protein", NATURE BIOTECHNOLOGY, vol. 40, 31 July 2022 (2022-07-31), pages 1132 - 1142, XP037903041, DOI: 10.1038/s41587-022-01278-2 *

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