WO2024032020A1 - 一种增强型单体StayGold蛋白及其应用 - Google Patents

一种增强型单体StayGold蛋白及其应用 Download PDF

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WO2024032020A1
WO2024032020A1 PCT/CN2023/089027 CN2023089027W WO2024032020A1 WO 2024032020 A1 WO2024032020 A1 WO 2024032020A1 CN 2023089027 W CN2023089027 W CN 2023089027W WO 2024032020 A1 WO2024032020 A1 WO 2024032020A1
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protein
staygold
mutated
mutation
monomeric
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PCT/CN2023/089027
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French (fr)
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王峰
吴家权
陈倩
杨益虎
桂文君
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无锡佰翱得生物科学股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of recombinant protein technology, and specifically to an enhanced monomeric StayGold protein and its application.
  • fluorescent proteins are widely used as marker proteins and reporter proteins in many fields of life science research to study the organization and function of living systems.
  • green fluorescent protein GFP
  • GFP green fluorescent protein
  • EGFP enhanced photostable green fluorescent protein
  • the purpose of this disclosure is to overcome the shortcomings of the existing technology and provide an enhanced monomeric StayGold protein and its application.
  • the present disclosure provides an enhanced monomeric StayGold protein.
  • the sequence of the enhanced monomeric StayGold protein is a mutant sequence of the wild-type two-body StayGold protein, and the mutation in the mutant sequence is the 137th sequence of the wild-type two-body StayGold protein.
  • At least one amino acid among asparagine (N) at position 140, glutamine (Q) at position 140, and tyrosine (Y) at position 187 is mutated into any amino acid.
  • the mutation of asparagine (N) at position 137 of the wild-type two-body StayGold protein is selected from the group consisting of N137A mutated to alanine (A), N137R mutated to arginine (R), N137G is glycine (G), N137H is mutated to histidine (H), N137I is mutated to isoleucine (I), N137L is mutated to leucine (L), and proline (P) is mutated N137P and N137S mutated to serine (S).
  • the mutation of asparagine (N) at position 137 of the wild-type two-body StayGold protein is N137A mutated to alanine (A).
  • the mutation of glutamine (Q) at position 140 of the wild-type disomeric StayGold protein is selected from the group consisting of Q140S mutated to serine (S), Q140A mutated to alanine (A), and mutated to glycine Q140G of (G), Q140H mutated to histidine (H), Q140I mutated to isoleucine (I), Q140L mutated to leucine (L), Q140P mutated to proline (P) and Q140Y mutated to tyrosine (Y).
  • the mutation of glutamine (Q) at position 140 of the wild-type disomeric StayGold protein is Q140S mutated to serine (S).
  • the mutation of tyrosine (Y) at position 187 of the wild-type disomeric StayGold protein is selected from the group consisting of Y187F mutated to phenylalanine (F), Y187A mutated to alanine (A), Y187N mutated to asparagine (N), Y187D mutated to aspartic acid (D), Y187I mutated to isoleucine (I), Y187L mutated to leucine (L), Y187L mutated to proline Y187P which is acid (P), Y187S which is mutated to serine (S) and Y187V which is mutated to valine (V).
  • the mutation of tyrosine (Y) at position 187 of the wild-type disomeric StayGold protein is Y187F, which is mutated to phenylalanine (F).
  • the mutation in the mutant sequence is one of the following four situations:
  • the mutation in the mutation sequence is a combined mutation of two positions at positions 137 and 140 of the wild-type two-body StayGold protein. In some more specific embodiments, the mutation in the mutation sequence is a combined mutation of two sites, N137A and Q140S, of the wild-type two-body StayGold protein.
  • the mutation in the mutation sequence is a combined mutation of two positions at positions 137 and 187 of the wild-type binary StayGold protein. In some more specific embodiments, the mutation in the mutation sequence is a combined mutation of two sites, N137A and Y187F, of the wild-type two-body StayGold protein.
  • the mutation in the mutation sequence is a combined mutation of two positions at positions 140 and 187 of the wild-type binary StayGold protein. In some more specific embodiments, the mutation in the mutation sequence is a combined mutation of two sites, Q140S and Y187F, of the wild-type disomeric StayGold protein.
  • the mutation in the mutation sequence is a combined mutation of three positions at positions 137, 140 and 187 of the wild-type two-body StayGold protein. In some more specific embodiments, the mutation in the mutation sequence is a combined mutation of three sites of N137A, Q140S and Y187F of the wild-type two-body StayGold protein.
  • the mutations in the mutated sequence are selected from any of the following:
  • amino acid sequence of the enhanced monomeric StayGold protein is shown in SEQ ID NO. 1. In other embodiments, the amino acid sequence of the enhanced monomeric StayGold protein is as shown in any one of SEQ ID NO. 3, 6-11.
  • the present disclosure also provides a polynucleotide encoding the above-mentioned enhanced monomeric StayGold protein.
  • sequence of the polynucleotide is set forth in SEQ ID NO. 2. In other embodiments, the sequence of the polynucleotide is set forth in SEQ ID NO. 4.
  • the present disclosure also provides a recombinant plasmid, which is an expression vector containing the above polynucleotide and capable of translating and expressing the above enhanced monomeric StayGold protein.
  • the expression vector is a pET28a vector.
  • the present disclosure also provides a protein expression system, which is the E. coli BL21 strain transformed into the above recombinant plasmid.
  • the present disclosure also provides a method for preparing the above-mentioned enhanced monomeric StayGold protein, which includes the following steps:
  • Step S1 Use gene synthesis technology to obtain the gene for the StayGold protein, construct the gene on the pET28a vector, and obtain the pET28a-6His-Strep II-TEV-GG-StayGold recombinant plasmid;
  • Step S2 Using the pET28a-6His-Strep II-TEV-GG-StayGold recombinant plasmid as a template, design saturated mutation primers for the amino acids that need to be mutated, perform PCR amplification, and obtain the target fragment of the monomeric StayGold gene. Constructed on the pET28a vector, the StayGold saturated mutation plasmid was obtained;
  • Step S3 Use the E. coli expression system to express the above saturated mutant protein, use fluorescence detection size exclusion chromatography (FSEC) to identify the monomeric StayGold protein in the saturated mutation, and use LC-MS to detect the molecular weight of the saturated mutant to identify the post-mutation probability. Higher potential amino acid sites;
  • FSEC fluorescence detection size exclusion chromatography
  • Step S4 Design site-directed mutation primers for the potential mutated amino acids screened by LC-MS, obtain the monomeric mutant protein according to step S2, and obtain the enhanced monomeric protein after verification using Ni-NTA affinity purified gel filtration chromatography. Body StayGold protein.
  • the present disclosure also provides an application of the above-mentioned enhanced monomeric StayGold protein in detecting protein labeling and expression localization.
  • This disclosure has the following beneficial effects: This disclosure provides for the first time a new monomeric StayGold protein, mStayGold.
  • This green fluorescent protein has strong thermal stability and fluorescence intensity, and its Tm is about 95°C, which is higher than commonly used in the market.
  • the Tm value of EGFP is 15°C higher, which is equivalent to the thermal stability of the two-body StayGold. Its fluorescence intensity is also 5 times higher than that of commonly used EGFP.
  • the nature of its monomer solves the problem of its wild-type disomic protein being fused with a target protein to track and locate the target protein due to its own aggregation state that affects the labeling and positioning functions. It has better application in guiding protein expression, cell positioning and other applications. Application scenarios.
  • Figure 1 shows the StayGold nickel column purification diagram
  • Figure 2 is the StayGold quality inspection chart
  • Figure 3 is the plate diagram after mStayGold saturation mutation
  • Figure 4 shows the rapid detection of mStayGold saturated mutation product FSEC
  • Figure 5 is a small purification diagram of mStayGold N137 saturated mutation sample
  • Figure 6 is a small purification diagram of mStayGold Q140 saturated mutation sample
  • Figure 7 shows the LC-MS detection chart of mStayGold N137 saturation mutation sample
  • Figure 8 shows the LC-MS detection chart of mStayGold Q140 saturated mutation sample
  • Figure 9 shows the LC-MS detection chart of mStayGold Y187 saturation mutation sample
  • Figure 10 shows the Tm value detection chart of mStayGold and other green fluorescent proteins
  • Figure 11 shows the fluorescence intensity detection chart of mStayGold and other green fluorescent proteins
  • Figure 12 shows the mStayGold site-directed single mutant protein nickel column purification diagram
  • Figure 13 is a picture of mStayGold site-specific single mutant protein aggregation status detection
  • Figure 14 shows the Tm value detection chart of mStayGold site-directed mutagen protein
  • Figure 15 shows the fluorescence intensity detection chart of mStayGold site-directed mutant protein
  • Figure 16 shows the mStayGold site-directed double mutant protein nickel column purification diagram
  • Figure 17 is a picture of mStayGold site-directed double mutant protein aggregation status detection
  • Figure 18 is a purification diagram of mStayGold (N137A, Q140S, Y187F) protein nickel column;
  • Figure 19 is a graph showing the detection of mStayGold (N137A, Q140S, Y187F) protein aggregation status.
  • This disclosure uses gene synthesis technology to obtain the disomic StayGold gene, and the gene is synthesized on the pET28a vector (Ubao Biotech, product number: VT1207). Transform the successfully constructed and correctly sequenced pET28a-6His-Strep II-TEV-GG-StayGold recombinant plasmid into BL21 (DE3) Escherichia coli competent cells (Biotech, Cat.
  • the molecular sieve buffer is 50mM Tris-HCl (pH 8.0) and 150mM NaCl.
  • the protein purified by molecular sieve has good uniformity and high purity. A small amount of samples were collected for quality testing. The results are shown in Figure 2.
  • the StayGold protein has high purity.
  • the LC-MS result shows that the molecular weight is 24760Da, which is very close to the theoretical molecular weight of StayGold of 24778Da.
  • the analysis The results of molecular sieve showed that the molecular weight of the protein was about 45kD, indicating that the StayGold protein was disomic in solution.
  • the mutated mStayGold gene is obtained by designing saturated mutation primers and using conventional molecular biology methods, and the gene is constructed on the pET28a vector.
  • the specific operation steps include:
  • the primers are shown in Table 1. The bold and underlined fonts are the corresponding amino acid sites that need to be mutated.
  • NNN represents 3 bases. is any amino acid, and its primer is a mixed primer.
  • the PCR product is connected to the pET28a vector using an in vitro ligation kit.
  • the ligated product is transformed into DH5 ⁇ Among the strains, the transformed bacterial solution was then spread on LB solid medium and cultured at 37°C overnight.
  • the plate after overnight culture was divided into four areas (Figure 3). Scrape the colonies from each area and place them into 5 mL of LB liquid culture medium. Cultivate overnight at 37°C. Collect the bacteria from each area for plasmid extraction.
  • N A/C/G/T.
  • the purification results of Q140 saturation mutation are similar to N137, as shown in Figure 6, and the molecular weight is close to the molecular weight of StayGold protein. It shows that the saturation mutation of any amino acid in N137, Q140 and Y187 screened by FSEC in this disclosure is a monomeric mStayGold protein.
  • the StayGold protein can be transformed from a dimer to a monomer.
  • the nickel column purified samples were subjected to LC-MS detection.
  • LC-MS detection results are shown in Figure 7.
  • the main molecular weight of the N137 saturated mutant protein sample is 27623 Da, which is very close to the molecular weight of the N137A mutation, indicating that the proportion of the N137A mutant in the saturated mutant sample is higher.
  • the StayGold protein can be transformed from a dimer to a monomer.
  • the samples purified by the nickel column were tested by LC-MS.
  • the LC-MS detection results are shown in Figure 8.
  • the main molecular weight of the Q140 saturated mutant protein sample is 27621 Da, which is very close to the molecular weight of the Q140S mutation, indicating that the proportion of Q140S mutant in the saturated mutant sample is higher.
  • the molecular weights corresponding to these peaks correspond to the potential mutants Q140A, Q140G, Q140H, Q140I, Q140L, Q140P, and Q140Y respectively.
  • the StayGold protein can be transformed from a dimer to a monomer.
  • the samples purified by the nickel column were tested by LC-MS.
  • the LC-MS detection results are shown in Figure 9.
  • the main molecular weight of the Y187 saturated mutant protein sample is 27600 Da, which is very close to the molecular weight of the Y187P mutation, indicating that the proportion of the Y187P mutant in the saturated mutant sample is higher.
  • the molecular weights corresponding to these peaks correspond to the potential mutants Y187F, Y187A, Y187N, Y187D, Y187I, Y187L, Y187S and Y187V respectively.
  • Tm values of TGP, EGFP and sfGFP are 71.95°C, 80.7°C and 87°C respectively; the Tm value of mStayGold is too high and exceeds the highest value detected by the instrument. According to its Tm curve It seems that its Tm value is about 95°C. It shows that the thermal stability of mStayGold of the present disclosure is better than that of green fluorescent eggs commonly used in the market, and has better application scenarios.
  • mStayGold N137A, mStayGold Q140S and mStayGold Y187F single mutations were randomly constructed.
  • the construction method was a conventional biological method.
  • the Tm values of the three single-mutated mStayGold are all too high, exceeding the highest value detected by the instrument. According to its Tm curve, the Tm value of mStayGold N137A is about 90°C.
  • the Tm values of mStayGold Q140S and mStayGold Y187F are about 95°C, and their Tm values are higher than those of EGFP and Tm values. It shows that the thermal stability of the single mutant monomer protein of mStayGold of the present disclosure is better than that of the green fluorescent eggs commonly used on the market, and has better application scenarios.
  • pET28a-6His-Strep II-TEV-GG-mStayGold N137A, Q140S
  • pET28a-6His-Strep II were constructed -TEV-GG-mStayGold (N137A, Y187F)
  • pET28a-6His-Strep II-TEV-GG-mStayGold Q140S, Y187F
  • the three recombinant plasmids that were successfully constructed and sequenced correctly were transformed into BL21 (DE3) Escherichia coli competent cells.
  • the strains were inoculated into 5 mL LB liquid medium and cultured at 37°C overnight.
  • the bacteria cultured overnight were inoculated at a ratio of 1:100.
  • Into 0.1L LB liquid culture medium culture at 37°C until the OD 600 of the bacterial solution is 0.6-0.8. Add 0.5mM IPTG and culture at 15°C overnight. Centrifuge at 5000rpm to collect the cells.
  • lysis buffer 50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol
  • a high-pressure homogenizer to crush the bacterial cells at a high speed of 16,000 rpm. Collect the supernatant by centrifugation.
  • affinity chromatography His FF to enrich and purify proteins.
  • lysis buffer to balance the His FF column. After hanging all cell supernatants on the column, use different gradients of imidazole solutions to elute, and collect the proteins eluted from different gradients of imidazole. SDS-PAGE was performed and the purification results are shown in Figure 16.
  • mStayGold N137A, Q140S
  • mStayGold N137A, Y187F
  • mStayGold Q140S, Y187F
  • the molecular weights of mStayGold(N137A,Q140S), mStayGold(N137A,Y187F) and mStayGold(Q140S,Y187F) proteins calculated by analytical molecular sieves are approximately 29, 19 and 24kD respectively, which is different from their theoretical molecular weights. 28kD is close, indicating that the three double mutant proteins are all monomeric mStayGold proteins.
  • the Tm values of the three double-mutated mStayGold are all too high, exceeding the highest value detected by the instrument. According to the Tm curve, the Tm value is about 95°C. Both are higher than EGFP and Tm values. It shows that the thermal stability of the double mutant monomer protein of mStayGold of the present disclosure is better than that of the green fluorescent eggs commonly used in the market, and has better application scenarios.
  • the fluorescence signals of the three proteins are all stronger than EGFP, mStayGold (N137A, Q140S), mStayGold (N137A, Y187F) and mStayGold (Q140S, Y187F), which are 1.7, 2.4 and 3.1 higher than EGFP respectively. times. It shows that the fluorescence intensity of the single mutant monomer protein of mStayGold disclosed in the present disclosure is better than that of the green fluorescent eggs commonly used in the market, and has a wider range of application scenarios.
  • mStayGold a recombinant plasmid using mStayGold (N137A, Q140S, Y187F) was constructed using conventional biological methods. Transform the successfully constructed and correctly sequenced pET28a-6His-Strep II-TEV-GG-mStayGold (N137A, Q140S, Y187F) recombinant plasmid into BL21 (DE3) E.
  • coli competent cells and graft the strain into 50 mL LB liquid medium Cultivate overnight at 37°C, add the bacteria cultured overnight into 1L LB liquid medium at a ratio of 1:100, and cultivate at 37°C until the bacterial liquid is When the OD 600 is 0.6-0.8, add 0.5mM IPTG and culture at 15°C overnight, and centrifuge at 5000rpm to collect the cells.
  • lysis buffer 50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol
  • lysis buffer 50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol
  • lysis buffer 50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol
  • Supernatant Use affinity chromatography His FF to enrich and purify proteins.
  • lysis buffer Before purification, use lysis buffer to balance the His FF column. After hanging all cell supernatants on the column, use different gradients of imidazole solutions to elute, and collect the proteins eluted from different gradients of imidazole. SDS-PAGE was performed and the purification results are shown in Figure 18.
  • mStayGold (N137A, Q140S, Y187F) proteins are all clearly expressed and have good purity. The yield can reach 193 mg/L, which is 2.5 times that of the disomic
  • the molecular sieve buffer is 50mM Tris-HCl (pH 8.0) and 150mM NaCl.
  • the results of the molecular sieve are shown in Figure 19.
  • the molecular weight of mStayGold (N137A, Q140S, Y187F) protein is about 22.7kD, and its theoretical molecular weight is 24.6kD, that is, the triple mutant is a monomeric mStayGold protein. This shows that the combined mutations of N137, Q140 and Y187 can also obtain monomeric mStayGold protein.
  • Tm values of mStayGold are all too high, exceeding the maximum value detected by the instrument. According to its Tm curve, the Tm value is about 90°C, and its Tm The value is higher than the EGFP and Tm values. It shows that the thermal stability of mStayGold (N137A, Q140S, Y187F) protein of the present disclosure is better than that of green fluorescent eggs commonly used on the market, and has better application scenarios.
  • 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.3 Amino acid sequence of combined mutant mStayGold (N137A, Q140S, Y187F) (artificial sequence)
  • SEQ ID NO.4 Nucleotide sequence encoding the combined mutant mStayGold (N137A, Q140S, Y187F) (artificial sequence)
  • SEQ ID NO.5 Amino acid sequence of wild-type disomeric StayGold protein (jellyfish, Cytaeis uchidae)
  • SEQ ID NO.6 Amino acid sequence of single mutant mStayGold (N137A) (artificial sequence)
  • SEQ ID NO.7 Amino acid sequence of single mutant mStayGold (Q140S) (artificial sequence)
  • SEQ ID NO.8 Amino acid sequence of single mutant mStayGold (Y187F) (artificial sequence)
  • SEQ ID NO.9 Double mutant mStayGold (N137A, Q140S) amino acid sequence (artificial sequence)
  • SEQ ID NO.10 Double mutant mStayGold (N137A, Y187F) amino acid sequence (artificial sequence)
  • SEQ ID NO.11 Amino acid sequence of double mutant mStayGold (Q140S, Y187F) (artificial sequence)

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Abstract

提供一种增强型单体StayGold蛋白及其应用,涉及重组蛋白技术领域，该增强型单体StayGold蛋白序列为野生型二体StayGold蛋白的突变序列，且所述突变序列中的突变为N137、Q140,Y187中至少一个氨基酸进行突变成任意氨基酸。所述新的单体的StayGold蛋白，mStayGold,该绿色荧光蛋白具有很强的热稳定性和荧光强度，其Tm约为95°C，比常用的EGFP的Tm值高出15°C,与二体的StayGold的热稳定相当，产量也高出2.5倍；其荧光强度也比常用EGFP的荧光强度高5倍。其单体的性质解决了其野生型二体蛋白在与目标蛋白融合进行追踪和定位目标蛋白时因自身聚集状态影响标记和定位的功能，在指导蛋白表达、细胞定位等应用中具有更好的应用场景。

Description

一种增强型单体StayGold蛋白及其应用
优先权和相关申请
本公开要求2022年08月09日提交的名称为“一种增强型单体StayGold蛋白及其应用”的中国专利申请202210949273.5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单体StayGold蛋白及其应用。
背景技术
如今，荧光蛋白(FPs)作为标记蛋白和报告蛋白被广泛应用于生命科学研究的诸多领域，以研究生命系统的组织和功能。其中绿色荧光蛋白(green fluorescent protein，GFP)是一类存在于水母、水媳等肠腔生物的应用最早的荧光蛋白。GFP广泛应用后，一系列相关的荧光蛋白产品已经被开发。虽然已经开发了大量明亮的绿色发射FPs，但大多数都不如增强型的光稳定绿色荧光蛋白(EGFP)，主要的原因在于当前很多FPs的光稳定性不好，限制了其应用，因此发现和设计出荧光强度和光稳定性产品对于荧光蛋白的应用非常重要。
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[1.Hirano，Masahiko et al.“A highly photostable and bright green fluorescent protein.”Nature biotechnology，10.1038/s41587-022-01278-2.25Apr.2022]。将荧光蛋白与目标蛋白进行融合，从而追踪、定位目标蛋白及检测其表达情况等是荧光蛋白较为广泛的应用。在这方面的应用中对于荧光蛋白的一个重要的考量标准就是荧光蛋白的单体性质，大部分荧光蛋白都容易形成低亲和力的二体或者多聚体，限制了它们与感兴趣的蛋白质产生融合的适用性。虽然说StayGold与其他常用荧光相比具有很好的光稳定性，但是StayGold属于一个二体蛋白，研究人员在使用其检测微管动态变化时还需要创建一个串联的二聚体，这样才能增加其光稳定性。如果开发一种单体的StayGold蛋白可以很好地解决其应用过程中的缺陷。
发明内容
本公开的目的在于克服现有技术的不足，提供了一种增强型单体StayGold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单体StayGold蛋白，该增强型单体StayGold蛋白序列为野生型二体StayGold蛋白的突变序列，且所述突变序列中的突变为将野生型二体StayGold蛋白的第137位的天冬酰胺(N)、第140位的谷氨酰胺(Q)、第187位的酪氨酸(Y)中至少一个氨基酸突变成任意氨基酸。
在一些实施方式中，野生型二体StayGold蛋白的第137位的天冬酰胺(N)的突变选自突变为丙氨酸(A)的N137A、突变为精氨酸(R)的N137R、突变为甘氨酸(G)的N137G、突变为组氨酸(H)的N137H、突变为异亮氨酸(I)的N137I、突变为亮氨酸(L)的N137L、突变为脯氨酸(P)的N137P和突变为丝氨酸(S)的N137S。
在一些具体实施方式中，野生型二体StayGold蛋白的第137位的天冬酰胺(N)的突变是突变为丙氨酸(A)的N137A。
在一些实施方式中，野生型二体StayGold蛋白的第140位的谷氨酰胺(Q)的突变选自突变为丝氨酸(S)的Q140S、突变为丙氨酸(A)的Q140A、突变为甘氨酸(G)的Q140G、突变为组氨酸(H)的Q140H、突变为异亮氨酸(I)的Q140I、突变为亮氨酸(L)的Q140L、突变为脯氨酸(P)的Q140P和突变为酪氨酸(Y)的Q140Y。
在一些具体实施方式中，野生型二体StayGold蛋白的第140位的谷氨酰胺(Q)的突变是突变为丝氨酸(S)的Q140S。
在一些实施方式中，野生型二体StayGold蛋白的第187位的酪氨酸(Y)的突变选自突变为苯丙氨酸(F)的Y187F、突变为丙氨酸(A)的Y187A、突变为天冬酰胺(N)的Y187N、突变为天冬氨酸(D)的Y187D、突变为异亮氨酸(I)的Y187I、突变为亮氨酸(L)的Y187L、突变为脯氨酸(P)的Y187P、突变为丝氨酸(S)的Y187S和突变为缬氨酸(V)的Y187V。
在一些具体实施方式中，野生型二体StayGold蛋白的第187位的酪氨酸(Y)的突变是突变为苯丙氨酸(F)的Y187F。
在一些实施方式中，所述突变序列中的突变为以下四种情形之一：
(1)对野生型二体StayGold蛋白的第137位和第140位两个位点组合突变；
(2)对野生型二体StayGold蛋白的第137位和第187位两个位点组合突变；
(3)对野生型二体StayGold蛋白的第140位和第187位两个位点组合突变；
(4)对野生型二体StayGold蛋白的第137位、第140位和第187位三个位点组合突变。
在一些具体实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的第137位和第140位两个位点组合突变。在一些更具体的实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的N137A和Q140S两个位点组合突变。
在一些具体实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的第137位和第187位两个位点组合突变。在一些更具体的实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的N137A和Y187F两个位点组合突变。
在一些具体实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的第140位和第187位两个位点组合突变。在一些更具体的实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的Q140S和Y187F两个位点组合突变。
在一些具体实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的第137位、第140位和第187位三个位点组合突变。在一些更具体的实施方式中，所述突变序列中的突变为对野生型二体StayGold蛋白的N137A、Q140S和Y187F三个位点组合突变。
在一些示例性的实施方式中，所述突变序列中的突变选自以下任一项：
(a)N137A；
(b)Q140S；
(c)Y187F；
(d)N137A和Q140S；
(e)N137A和Y187F；
(f)Q140S和Y187F；
(g)N137A、Q140S和Y187F。
在一些实施方式中，所述增强型单体StayGold蛋白的氨基酸序列如SEQ ID NO.1所示。在另一些实施方式中，所述增强型单体StayGold蛋白的氨基酸序列如SEQ ID NO.3,6～11中任一项所示。
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单体StayGold蛋白。
在一些实施方式中，所述多核苷酸的序列如SEQ ID NO.2所示。在另一些实施方式中，所述多核苷酸的序列如SEQ ID NO.4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单体StayGold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单体StayGold蛋白的制备方法，包括以下步骤：
步骤S1：利用基因合成技术获得StayGold蛋白的基因，将该基因构建在pET28a载体上，获得pET28a-6His-Strep II-TEV-GG-StayGold重组质粒；
步骤S2：以pET28a-6His-Strep II-TEV-GG-StayGold重组质粒作为模板，设计需要进行突变的氨基酸的饱和突变引物，进行PCR扩增，获得单体的StayGold基因的目的片段，将目的片段构建在pET28a载体上，获得StayGold饱和突变质粒；
步骤S3：利用大肠杆菌表达系统表达上述饱和突变体蛋白，利用荧光检测尺寸排除色谱(FSEC)鉴定出饱和突变中的单体StayGold蛋白，使用LC-MS检测饱和突变体的分子量鉴定出突变后概率较高的潜在氨基酸位点；
步骤S4：设计由LC-MS筛选出的潜在突变后氨基酸的定点突变引物，按照步骤S2获得单体突变蛋白，使用Ni-NTA亲和纯化的凝胶过滤层析验证后获得所述增强型单体StayGold蛋白。
本公开还提供一种上述增强型单体StayGold蛋白在检测蛋白的标记、表达定位中的应用。
本公开具有如下有益效果：本公开首次提供了一种新的单体的StayGold蛋白，mStayGold，该绿色荧光蛋白具有很强的热稳定性和荧光强度，其Tm约为95℃，比市场上常用的EGFP的Tm值高出15℃，与二体的StayGold的热稳定相当，其荧光强度也比常用EGFP的荧光强度高5倍。其单体的性质解决了其野生型二体蛋白在与目标蛋白融合进行追踪和定位目标蛋白时因自身聚集状态影响标记和定位的功能，在指导蛋白表达、细胞定位等应用中具有更好的应用场景。
附图说明
图1为StayGold镍柱纯化图；
图2为StayGold质量检测图；
图3为mStayGold饱和突变后平板图；
图4为mStayGold饱和突变产物FSEC快速检图；
图5为mStayGold N137饱和突变样品的小量纯化图；
图6为mStayGold Q140饱和突变样品的小量纯化图；
图7为mStayGold N137饱和突变样品的LC-MS检测图；
图8为mStayGold Q140饱和突变样品的LC-MS检测图；
图9为mStayGold Y187饱和突变样品的LC-MS检测图
图10为mStayGold与其他绿色荧光蛋白Tm值检测图；
图11为mStayGold与其他绿色荧光蛋白荧光强度检测图；
图12为mStayGold定点单突变蛋白镍柱纯化图；
图13为mStayGold定点单突变蛋白聚集状态检测图；
图14为mStayGold定点突变蛋白Tm值检测图；
图15为mStayGold定点突变蛋白荧光强度检测图；
图16为mStayGold定点双突变蛋白镍柱纯化图；
图17为mStayGold定点双突变蛋白聚集状态检测图；
图18为mStayGold(N137A，Q140S，Y187F)蛋白镍柱纯化图；
图19为mStayGold(N137A，Q140S，Y187F)蛋白聚集状态检测图。
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 mStayGold蛋白表达及纯化
2.1.1 StayGold质粒构建及表达
本公开用基因合成技术获得二体的StayGold的基因，其基因是合成在pET28a载体(优宝生物，货号：VT1207)上。将构建成功且测序正确的pET28a-6His-Strep II-TEV-GG-StayGold重组质粒转化BL21(DE3)大肠杆菌感受态细胞(唯地生物，货号：EC1002)，将菌株接种至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。
2.1.2 StayGold重组蛋白镍柱纯化
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)，500mM NaCl，5％甘油)，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1所示，StayGold蛋白均明显表达且纯度较好且产量高达78.4mg/L。
2.1.3 StayGold重组蛋白分子筛纯化及质量检测
收集镍柱纯化后的蛋白进行分子筛纯化，分子筛缓冲液为50mM Tris-HCl(pH 8.0)，150mM NaCl。分子筛纯化后的蛋白均一性好且纯度高，收集少量样品进行质量检测，结果如图2所示，StayGold蛋白纯度高，LC-MS结果显示分子量为24760Da与StayGold的理论分子量24778Da非常接近，且分析型分子筛的结果显示蛋白分子量约为45kD，说明StayGold蛋白在溶液状态下为二体。
2.2 mStayGold质粒构建
本公开通过设计饱和突变引物，使用常规的分子生物学手段获得突变后的mStayGold基因，将其基因构建在pET28a载体上，具体操作步骤包括：
分别设计6个突变氨基酸N137、E138、Q140、Y187、W189、R191的饱和突变引物，其引物见表1，其中加粗划线字体为对应的需要突变氨基酸位点，NNN代表3个碱基可以为任意氨基酸，其引物为混合引物，以StayGold的基因作为模板，按照常规PCR手段进行PCR扩增，PCR后的产物使用体外连接试剂盒将PCR产物与pET28a载体进行连接，连接后的产物转化DH5α菌株中，之后将转化后的菌液涂布在LB固体培养基上，37℃，过夜培养。
将过夜培养后的平板分成四个区域(图3)。把每个区域上的菌落都刮下来分别接到5mL LB液体培养基中，37℃过夜培养，分别收集每个区域的菌体进行质粒抽提。
表1.mStayGold蛋白饱和突变引物
其中，N＝A/C/G/T。
2.3 mStayGold重组质粒小量表达
将抽提后的重组质粒按照常规分子生物学手段转化BL21(DE3)大肠杆菌感受态细胞，分别挑取单克隆菌斑至5mL LB液体培养基中，37℃培养，待菌液OD600至0.6-0.8时，加入0.5mM IPTG，15℃诱导16个小时。分别收集菌体，用裂解buffer将菌体吹匀后，使用超声破碎仪超声10秒，将超声后的样品用16000rpm高速离心10分钟收集上清。
2.3.1 FSEC快速检测蛋白聚集状态
取部分上清稀释至0.1mg/ml，稀释后的上清取100μL样品进行FSEC系统检测，检测结果如图4所示，N137、Q140和Y187三个氨基酸的饱和突变产物在FSEC上均只有少量高聚峰和单一的分子量约为19kDa的单峰，StayGold单体的理论分子量为27kD，初步判断为单体的mStayGold蛋白；E138、W189和R191三个氨基酸的饱和突变产物在FSEC上除了少量高聚峰和比例较高的19kDa的单峰外还有部分分子量约为41kD的峰。
这表明，N137、Q140和Y187饱和突变成任意氨基酸大概率都能获得单体mStayGold蛋白，而E138、W189和R191三个氨基酸饱和突变成某些氨基酸可以获得单体mStayGold蛋白或者突变成任意氨基酸都不能够获得完全的mStayGold蛋白。
2.3.2小量纯化验证FSEC筛选的准确性
为了进一步验证FSEC筛选的N137、Q140和Y187的单体mStayGold是否为目标蛋白，我们将N137和Q140饱和突变产物的上清液进行了镍柱纯化，纯化的具体操作是将上清液与少量Ni胶质在4℃孵育30min，16000rpm高速离心5分钟去除上清液并取少量进行SD-PAGE检测，再在反应液中加入200μL含500mM咪唑的裂解缓冲液，16000rpm高速离心5分钟去除收集上清液并取少量进行SDS-PAGE检测。N137饱和突变样品的小量纯化结果如图5所示，4个区域蛋白均可以洗脱，且分子量与目标蛋白接近，说明FSEC筛选出的～19kD的峰为mStayGold蛋白。
Q140饱和突变的纯化结果与N137相似，如图6所示，分子量与StayGold蛋白分子量接近。说明本公开通过FSEC筛选出的N137、Q140和Y187中任一氨基酸的饱和突变均为单体的mStayGold蛋白。
2.4 N137饱和突变位点验证
为了进一步确认N137位氨基酸突变成何种氨基酸可以实现StayGold蛋白从二体变成单体。将镍柱纯化后的样品进行LC-MS检测，通过检测N137饱和突变蛋白样品的分子量，对照突变后氨基酸的理论分子量推断出N137突变后潜在的氨基酸。LC-MS的检测结果如图7所示，N137饱和突变蛋白样品的主要分子量为27623Da，与N137A突变的分子量非常接近，说明饱和突变样品中N137A突变体的比例较高。
此外，在27623Da附近还有一些峰，这些峰对应的分子量分别对应的潜在突变体为N137R、N137G、N137H、N137I、N137L、N137P和N137S。
2.5 Q140饱和突变位点验证
为了确认Q140位氨基酸突变成何种氨基酸可以实现StayGold蛋白从二体变成单体。将镍柱纯化后的样品进行LC-MS检测，通过检测Q140饱和突变蛋白样品的分子量，对照突变后氨基酸的理论分子量推断出Q140突变后潜在的氨基酸。LC-MS的检测结果如图8所示，Q140饱和突变蛋白样品的主要分子量为27621Da，与Q140S突变的分子量非常接近，说明饱和突变样品中Q140S突变体的比例较高。此外，在27621Da附近还有一些峰，这些峰对应的分子量分别对应的潜在突变体为Q140A、Q140G、Q140H、Q140I、Q140L、Q140P、Q140Y。
2.6 Y187突变位点验证
为了确认Y187位氨基酸突变成何种氨基酸可以实现StayGold蛋白从二体变成单体。将镍柱纯化后的样品进行LC-MS检测，通过检测Y187饱和突变蛋白样品的分子量，对照突变后氨基酸的理论分子量推断出Y187突变后潜在的氨基酸。LC-MS的检测结果如图9所示，Y187饱和突变蛋白样品的主要分子量为27600Da，与Y187P突变的分子量非常接近，说明饱和突变样品中Y187P突变体的比例较高。此外，在27600Da附近还有一些峰，这些峰对应的分子量分别对应的潜在突变体为Y187F、Y187A、Y187N、Y187D、Y187I、Y187L、Y187S和Y187V。
2.7 mStayGold饱和突变蛋白热稳定检测
为了研究单体的mStayGold蛋白的热稳定性，选择小量纯化后的N137的饱和突变蛋白将其与市场上常用的热稳定好的几种绿色荧光蛋白：超折叠绿色荧光蛋白(sfGFP)、增强绿色荧光蛋白(EGFP)和热稳定性的绿色荧光蛋白(TGP)进行Tm值的比较。具体的实验操作如下：
取20μL上述4种蛋白(N137饱和突变产物(图10中标记为mStayGold)、sfGFP蛋白、EGFP蛋白和TGP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10所示，TGP、EGFP和sfGFP的Tm值分别为71.95℃、80.7℃和87℃；mStayGold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℃。表明本公开的mStayGold的热稳定性比市场上常用的绿色荧光蛋要好，具有更好的应用场景。
2.8 mStayGold饱和突变蛋白荧光强度检测
为了研究单体的mStayGold蛋白的荧光强度，选择小量纯化后的N137的饱和突变蛋白将其与市场上常用的热稳定好的几种绿色荧光蛋白：超折叠绿色荧光蛋白(sfGFP)、增强绿色荧光蛋白(EGFP)和热稳定性的绿色荧光蛋白(TGP)进行Tm值的比较。具体的实验操作如下：
分别取10μL 4种不同的荧光蛋白(N137饱和突变产物(图11中标记为mStayGold)、sfGFP蛋白、EGFP蛋白和TGP蛋白)于384孔的反应板中，加入10μL反应缓冲液(20mM Tris-HCl pH 7.0，150mM NaCl)，用全波长扫描仪进行读数，设置激发波长为488nm，发射波长为510nm。实验数据如图11所示，mStayGold呈现出最高的荧光信号，分别比 sfGFP、EGFP和TGP分别高出3、5.3和3.3倍。表明本公开的mStayGold的荧光强度比市场上常用的绿色荧光蛋要好，具有更广泛的应用场景。
2.9 mStayGold定点单突变蛋白稳定性及荧光强度检测
2.9.1 mStayGold定点单突变蛋白表达
通过LC-MS检测，推测出mStayGold潜在的突变体后，随机构建了mStayGold N137A、mStayGold Q140S和mStayGold Y187F单突变，其构建方法为常规生物学方法。将构建成功且测序正确的pET28a-6His-Strep II-TEV-GG-mStayGold N137A、pET28a-6His-Strep II-TEV-GG-mStayGold Q140S和pET28a-6His-Strep II-TEV-GG-mStayGold Y187F重组质粒转化BL21(DE3)大肠杆菌感受态细胞，将菌株接中至5mL 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0.1L 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。
2.9.2 mStayGold定点单突变蛋白镍柱纯化
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)，500mM NaCl，5％甘油)，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12所示，mStayGold N137A、mStayGold Q140S和mStayGold Y187F蛋白均明显表达且纯度较好。
2.9.3 mStayGold定点单突变蛋白聚集状态检测
为了进一步明确mStayGold N137A和mStayGold Q140S蛋白的聚集状态，分别收集镍柱纯化后的蛋白进行分子筛纯化，分子筛缓冲液为50mM Tris-HCl(pH 8.0)，150mM NaCl。分子筛的结果如图13所示，mStayGold N137A、mStayGold Q140S和mStayGold Y187F蛋白通过分析型分子筛计算出的分子量分别约为29、34和22kD，与其理论分子量28kD接近，说明三个突变体均为单体mStayGold蛋白。
2.9.4 mStayGold定点单突变蛋白热稳定检测
取20μL上述3种mStayGold N137A、mStayGold Q140S和mStayGold Y187F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14所示，3种单突变的mStayGold的Tm值均太高，超出了仪器检测的最高值，根据其Tm的曲线来看，mStayGold N137A的Tm值约为90℃，mStayGold Q140S和mStayGold Y187F的Tm值约为95℃，其Tm值均比EGFP和Tm值高。表明本公开的mStayGold的单突变单体蛋白的热稳定性比市场上常用的绿色荧光蛋要好，具有更好的应用场景。
2.9.5 mStayGold定点单突变蛋白荧光强度检测
分别取10μL 3种mStayGold N137A、mStayGold Q140S和mStayGold Y187F蛋白于384孔的反应板中，加入10μL反应缓冲液(20mM Tris-HCl pH 7.0，150mM NaCl)，用全波长扫描仪进行读数，设置激发波长为488nm，发射波长为510nm。实验数据如图15所示，3种蛋白的荧光信号都比EGFP强，mStayGold N137A、mStayGold Q140S和mStayGold Y187F，分别比EGFP高出1.3、3.5和2.2倍。表明本公开的mStayGold的单突变单体蛋白的荧光强度比市场上常用的绿色荧光蛋要好，具有更广泛的应用场景。
2.10 mStayGold定点双突变蛋白稳定性及荧光强度检测
2.10.1 mStayGold定点双突变蛋白表达
在验证了mStayGold N137A、mStayGold Q140S和mStayGold Y187F单突变蛋白具有高稳定性及高荧光强度后，构建了pET28a-6His-Strep II-TEV-GG-mStayGold(N137A,Q140S)、pET28a-6His-Strep II-TEV-GG-mStayGold(N137A,Y187F)和pET28a-6His-Strep II-TEV-GG-mStayGold(Q140S，Y187F)3个双突变蛋白表达质粒进行验证，其构建方法为常规生物学方法。将构建成功且测序正确的3个重组质粒转化BL21(DE3)大肠杆菌感受态细胞，将菌株接中至5mL 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0.1L 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。
2.10.2 mStayGold定点双突变蛋白镍柱纯化
将收集的菌体进行称重，按照1:10比例加入相应体积的裂解缓冲液(50mM Tris-HCl(pH 7.5)，500mM NaCl，5％甘油)，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16所示，mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S，Y187F)蛋白均明显表达且纯度较好。
2.10.3 mStayGold定点双突变蛋白聚集状态检测
为了进一步明确mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S，Y187F)蛋白的聚集状态，分别收集镍柱纯化后的蛋白进行分子筛纯化，分子筛缓冲液为50mM Tris-HCl(pH 8.0)，150mM NaCl。分子筛的结果如图17所示，mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S,Y187F)蛋白通过分析型分子筛计算出的分子量分别约为29、19和24kD，与其理论分子量28kD接近，说明三个双突变体蛋白均为单体mStayGold蛋白。
2.10.4 mStayGold定点双突变蛋白热稳定检测
取20μL上述3种mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S,Y187F)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14所示，3种双突变的mStayGold的Tm值均太高，超出了仪器检测的最高值，根据其Tm的曲线来看，Tm值约为95℃，其Tm值均比EGFP和Tm值高。表明本公开的mStayGold的双突变单体蛋白的热稳定性比市场上常用的绿色荧光蛋要好，具有更好的应用场景。
2.10.5 mStayGold定点双突变蛋白荧光强度检测
分别取10μL 3种mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S,Y187F)蛋白于384孔的反应板中，加入10μL反应缓冲液(20mM Tris-HCl pH 7.0，150mM NaCl)，用全波长扫描仪进行读数，设置激发波长为488nm，发射波长为510nm。实验数据如图15所示，3种蛋白的荧光信号都比EGFP强，mStayGold(N137A,Q140S)、mStayGold(N137A,Y187F)和mStayGold(Q140S,Y187F)，分别比EGFP高出1.7、2.4和3.1倍。表明本公开的mStayGold的单突变单体蛋白的荧光强度比市场上常用的绿色荧光蛋要好，具有更广泛的应用场景。
2.11 mStayGold组合突变蛋白表达及纯化
2.11.1 mStayGold(N137A，Q140S，Y187F)蛋白表达
为了进一步明确N137、Q140和Y187组合突变后，是否还能获得单体的mStayGold蛋白，利用常规生物学方法构建了用mStayGold(N137A，Q140S，Y187F)重组质粒。将构建成功且测序正确的pET28a-6His-Strep II-TEV-GG-mStayGold(N137A，Q140S，Y187F)重组质粒转化BL21(DE3)大肠杆菌感受态细胞，将菌株接中至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。
2.11.2 mStayGold(N137A，Q140S，Y187F)蛋白纯化
将菌体进行称重后按照1:10比例加入相应体积的裂解缓冲液(50mM Tris-HCl(pH 7.5)，500mM NaCl，5％甘油)，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18所示，mStayGold(N137A，Q140S，Y187F)蛋白均明显表达且纯度较好，产量可达193mg/L，是二体的2.5倍。
2.11.3 mStayGold(N137A，Q140S，Y187F)蛋白聚集状态检测
收集镍柱纯化后的蛋白进行分子筛纯化，分子筛缓冲液为50mM Tris-HCl(pH 8.0)，150mM NaCl。分子筛的结果如图19所示，mStayGold(N137A，Q140S，Y187F)蛋白的分子量约为22.7kD，其理论分子量为24.6kD，即三突变体为单体mStayGold蛋白。说明N137、Q140和Y187的组合突变也能获得单体的mStayGold蛋白。
2.11.4 mStayGold(N137A，Q140S，Y187F)蛋白热稳定检测
取20μL上述mStayGold(N137A，Q140S，Y187F)蛋白加到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14所示，mStayGold(N137A，Q140S，Y187F)Tm值均太高，超出了仪器检测的最高值，根据其Tm的曲线来看，Tm值约为90℃，其Tm值比EGFP和Tm值高。表明本公开的mStayGold(N137A，Q140S，Y187F)蛋白的热稳定性比市场上常用的绿色荧光蛋要好，具有更好的应用场景。
2.11.5 mStayGold(N137A，Q140S，Y187F)蛋白荧光强度检测
分别取10μL mStayGold(N137A，Q140S，Y187F)蛋白于384孔的反应板中，加入10μL反应缓冲液(20mM Tris-HCl pH 7.0，150mM NaCl)，用全波长扫描仪进行读数，设置激发波长为488nm，发射波长为510nm。实验数据如图15所示，mStayGold(N137A，Q140S，Y187F)的荧光信号都比EGFP高出1.7、2.4和2.8倍。表明本公开的mStayGold的三突变单体蛋白的荧光强度比市场上常用的绿色荧光蛋要好，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(人工序列)
SEQ ID NO.3：组合突变体mStayGold(N137A，Q140S，Y187F)氨基酸序列(人工序列)
SEQ ID NO.4：编码组合突变体mStayGold(N137A，Q140S，Y187F)的核苷酸序列(人工序列)
SEQ ID NO.5：野生型二体StayGold蛋白的氨基酸序列(水母，Cytaeis uchidae)
SEQ ID NO.6：单突变体mStayGold(N137A)氨基酸序列(人工序列)
SEQ ID NO.7：单突变体mStayGold(Q140S)氨基酸序列(人工序列)
SEQ ID NO.8：单突变体mStayGold(Y187F)氨基酸序列(人工序列)
SEQ ID NO.9：双突变体mStayGold(N137A，Q140S)氨基酸序列(人工序列)
SEQ ID NO.10：双突变体mStayGold(N137A，Y187F)氨基酸序列(人工序列)
SEQ ID NO.11：双突变体mStayGold(Q140S，Y187F)氨基酸序列(人工序列)
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 (14)

  1. 一种增强型单体StayGold蛋白，其特征在于，所述增强型单体StayGold蛋白的序列为野生型二体StayGold蛋白的突变序列，且所述突变序列中的突变为将野生型二体StayGold蛋白的第137位的天冬酰胺(N)、第140位的谷氨酰胺(Q)、第187位的酪氨酸(Y)中至少一个氨基酸突变成任意氨基酸。
  2. 根据权利要求1所述的增强型单体StayGold蛋白，其特征在于，野生型二体StayGold蛋白的第137位的天冬酰胺(N)的突变选自突变为丙氨酸(A)的N137A、突变为精氨酸(R)的N137R、突变为甘氨酸(G)的N137G、突变为组氨酸(H)的N137H、突变为异亮氨酸(I)的N137I、突变为亮氨酸(L)的N137L、突变为脯氨酸(P)的N137P和突变为丝氨酸(S)的N137S。
  3. 根据权利要求1所述的增强型单体StayGold蛋白，其特征在于，野生型二体StayGold蛋白的第140位的谷氨酰胺(Q)的突变选自突变为丝氨酸(S)的Q140S、突变为丙氨酸(A)的Q140A、突变为甘氨酸(G)的Q140G、突变为组氨酸(H)的Q140H、突变为异亮氨酸(I)的Q140I、突变为亮氨酸(L)的Q140L、突变为脯氨酸(P)的Q140P和突变为酪氨酸(Y)的Q140Y。
  4. 根据权利要求1所述的增强型单体StayGold蛋白，其特征在于，野生型二体StayGold蛋白的第187位的酪氨酸(Y)的突变选自突变为苯丙氨酸(F)的Y187F、突变为丙氨酸(A)的Y187A、突变为天冬酰胺(N)的Y187N、突变为天冬氨酸(D)的Y187D、突变为异亮氨酸(I)的Y187I、突变为亮氨酸(L)的Y187L、突变为脯氨酸(P)的Y187P、突变为丝氨酸(S)的Y187S和突变为缬氨酸(V)的Y187V。
  5. 根据权利要求1所述的增强型单体StayGold蛋白，其特征在于，所述突变序列中的突变为以下四种情形之一：
    (1)对野生型二体StayGold蛋白的第137位和第140位两个位点组合突变；
    (2)对野生型二体StayGold蛋白的第137和第187位两个位点组合突变；
    (3)对野生型二体StayGold蛋白的第140和第187位两个位点组合突变；
    (4)对野生型二体StayGold蛋白的第137、第140位和第187位三个位点组合突变。
  6. 根据权利要求1-5任一项所述的增强型单体StayGold蛋白，其特征在于，所述突变序列中的突变选自以下任一项：
    (a)N137A；
    (b)Q140S；
    (c)Y187F；
    (d)N137A和Q140S；
    (e)N137A和Y187F；
    (f)Q140S和Y187F；
    (g)N137A、Q140S和Y187F。
  7. 根据权利要求1所述的增强型单体StayGold蛋白，其特征在于，所述增强型单体StayGold蛋白的氨基酸序列如SEQ ID NO.1所示；或，
    所述增强型单体StayGold蛋白的氨基酸序列如SEQ ID NO.3,6～11中任一项所示；
    优选地，所述增强型单体StayGold蛋白的氨基酸序列如SEQ ID NO.1所示。
  8. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1-7任一项所述的增强型单体StayGold蛋白。
  9. 根据权利要求8所述的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO.2所示；或，所述多核苷酸的序列如SEQ ID NO.4所示；
    优选地，所述多核苷酸的序列如SEQ ID NO.2所示。
  10. 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8-9任一项所述的多核苷酸且能够翻译表达出如权利要求1-7任一项所述增强型单体StayGold蛋白的表达载体。
  11. 根据权利要求10所述的一种重组质粒，其特征在于，所述表达载体为pET28a载体。
  12. 一种蛋白表达系统，其特征在于，所述蛋白表达系统为转入权利要求11所述重组质粒的大肠杆菌BL21菌株。
  13. 一种如权利要求1-7任一项所述的增强型单体StayGold蛋白的制备方法，其特征在于，包括以下步骤：
    步骤S1：利用基因合成技术获得StayGold蛋白的基因，将该基因构建在pET28a载体上，获得pET28a-6His-Strep II-TEV-GG-StayGold重组质粒；
    步骤S2：以pET28a-6His-Strep II-TEV-GG-StayGold重组质粒作为模板，设计需要进行突变的氨基酸的饱和突变引物，进行PCR扩增，获得单体的StayGold基因的目的片段，将目的片段构建在pET28a载体上，获得StayGold饱和突变质粒；
    步骤S3：利用大肠杆菌表达系统表达上述饱和突变体蛋白，利用荧光检测尺寸排除色谱FSEC鉴定出饱和突变中的单体StayGold蛋白，使用LC-MS检测饱和突变体的分子量鉴定出突变后概率较高的潜在氨基酸位点；
    步骤S4：设计由LC-MS筛选出的潜在突变后氨基酸的定点突变引物，按照步骤S2获得单体突变蛋白，使用Ni-NTA亲和纯化的凝胶过滤层析验证后获得所述增强型单体StayGold蛋白。
  14. 一种如权利要求1-7任一项所述的增强型单体StayGold蛋白在检测蛋白的标记、表达定位中的应用。
PCT/CN2023/089027 2022-08-09 2023-04-18 一种增强型单体StayGold蛋白及其应用 WO2024032020A1 (zh)

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