WO2023036210A1 - 一种含非天然氨基酸蛋白的制备方法 - Google Patents

一种含非天然氨基酸蛋白的制备方法 Download PDF

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WO2023036210A1
WO2023036210A1 PCT/CN2022/117667 CN2022117667W WO2023036210A1 WO 2023036210 A1 WO2023036210 A1 WO 2023036210A1 CN 2022117667 W CN2022117667 W CN 2022117667W WO 2023036210 A1 WO2023036210 A1 WO 2023036210A1
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trna
unnatural amino
amino acids
erf1
mutated
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夏青
史宁宁
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北京大学
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  • the invention belongs to the field of biopharmaceuticals, and in particular relates to a method for preparing a protein containing unnatural amino acids, in particular to a method for simultaneously efficiently inserting three different unnatural amino acids into a protein at a fixed point in a mammalian cell.
  • Unnatural amino acids based on different systems have been discovered and applied in research work, including tyrosine system, pyrrolysine system, and phenylalanine system, greatly enriching the selection of unnatural amino acid structures; and more and more species, including Escherichia coli, mammalian cells, yeast, insect cells, etc., can insert unnatural amino acids, which also lays the foundation for the wide application of this technology; in terms of insertion methods, from the earliest amber stop codon , to other stop codons, quadruple codons, rare codons, and even optimized special ribosomes, etc., providing more options for insertion methods.
  • tRNAs that recognize orthogonal codons and the corresponding high-specificity aaRS that can load unnatural amino acids onto tRNAs are required, so as to realize the synthesis of unnatural amino acids.
  • Orthogonal system needs to meet: a unique new codon, tRNA that can recognize the new codon, and aaRS that can be coupled to it; and, the aaRs/tRNA combination must be orthogonal to the endogenous aaRS/tRNA combination (that is, non-crossover ) to ensure the accuracy of unnatural amino acid incorporation.
  • Protein drugs including antibodies, are the fastest-growing, most profitable, and most competitive research and development field in the world. However, almost all protein drugs have the following major defects in common: 1It is easily degraded by proteases in the body, resulting in a short half-life; 2The molecular weight is large and the immunogenicity is strong, and the body injection induces the production of neutralizing antibodies in the body, resulting in a continuous decrease in drug efficacy; 3It cannot be modified at a fixed point, and lacks systematic research methods for structure-activity relationships like small molecule drugs. These bottlenecks plague the overall development of protein medicines, especially in terms of quality control and efficacy.
  • Green Fluorescent Protein the most commonly used reporter gene and a powerful tool to indicate unnatural amino acid insertions, consists of 238 amino acids.
  • This application uses the green fluorescent protein GFP as an example to illustrate the technical background of the present invention, but the following content should not be understood as an acknowledgment of the prior art, nor can it be considered that the present invention is only applicable to GFP protein.
  • unnatural amino acid protein modification at the gene level can only be modified at a single site, one unnatural amino acid, and it is impossible to achieve multiple site-specific modification of a protein, or modification of multiple proteins with different unnatural amino acids.
  • the problem has become a bottleneck in the application of gene codon expansion technology in the development of protein drugs. Therefore, how to apply the exogenous gene codon system to mammalian cells, improve the modification efficiency of non-natural amino acid residue polypeptides in mammalian cells, and realize simultaneous specific modification at multiple sites of a protein, or multiple Simultaneous expression of proteins with different unnatural amino acid residues is a technical problem to be solved urgently in this field.
  • the purpose of the present invention is to provide a method for simultaneously and efficiently inserting three different unnatural amino acids into a protein at a specific site in mammalian cells.
  • the invention provides a method for expressing recombinant proteins containing unnatural amino acids in animal cells.
  • aminoacyl-tRNA synthetases and corresponding tRNAs aminoacyl-tRNAs that recognize stop codons are constructed, and three aminoacyl-tRNAs are screened to obtain three A genetic code extension system.
  • the three gene codon expansion systems have high read-through efficiency and strong mutual compatibility for stop codons, so that three unnatural amino acids can be inserted into one foreign protein, or three different amino acids can be inserted into three foreign proteins in the same cell. unnatural amino acids.
  • the release factor eRF1 was also mutated to weaken its interaction with the stop codon.
  • the GFP protein modified at three sites was amplified and expressed, and a series of verifications such as protein purification and LC-MS mass spectrometry were performed to obtain a high-purity multi-site specific modified protein.
  • the present invention provides a kind of gene codon expansion composition, comprising:
  • the mutated tRNA anticodon loop is mutated into a complementary sequence of a stop codon
  • aminoacyl-tRNA synthetase can combine mutant tRNA to unnatural amino acid to produce aminoacyl-tRNA.
  • the gene codon expansion composition of the present invention is characterized in that:
  • aminoacyl-tRNA synthetase is selected from Pyl-tRNA synthetase (PylRS), Leu-tRNA synthetase (LeuRS), Tyr-tRNA synthetase (TyrRS);
  • the mutated tRNA is selected from tRNA Pyl , tRNA Leu , tRNA Tyr .
  • tRNAPyl and PylRS are from Methanosarcina mazei or Methanosarcina barkeri; tRNALeu and LeuRS are from Escherichia coli; tRNATyr and TyrRS from E. coli.
  • the gene codon expansion composition of the present invention includes one or more selected from the following combinations of aminoacyl-tRNA synthetase and mutant tRNA
  • the gene codon expansion composition of the present invention comprises
  • An aminoacyl-tRNA synthetase selected from any one of EcLeuRs and tRNA EcLeuUCA , EcLeuRs and tRNA EcLeuCUA , EcLeuRs and tRNA EcLeuUUA in combination with a mutant tRNA;
  • aminoacyl-tRNA synthetase selected from any one of OMeYRs and tRNA EcTyr UCA , OMeYRs and tRNA EcTyr CUA , OMeYRs and tRNA EcTyr UUA in combination with the mutant tRNA.
  • the gene codon expansion composition of the present invention includes the combination of three groups of aminoacyl-tRNA synthetases MmPylRs and tRNA MmPyl UCA , OMeYRs and tRNA EcTyr UUA , and EcLeuRs and tRNA EcLeu CUA and mutant tRNA.
  • any one of the aforementioned gene codon expansion compositions of the present invention is characterized in that it also includes unnatural amino acids, and the unnatural amino acids are preferably NAEK, Anap, and pAcF.
  • the present invention provides a method for expressing recombinant proteins containing unnatural amino acids in animal cells, comprising:
  • step (3) introducing the recombinant protein expression cassette in step (1) into the animal cell transformed in step (2) to prepare the recombinant animal cell;
  • the method for expressing a recombinant protein containing unnatural amino acids in animal cells according to the present invention is characterized in that the amino acid sequence of the recombinant protein contains one, two or three unnatural amino acids.
  • the method for expressing recombinant proteins containing unnatural amino acids in animal cells according to the present invention is characterized in that one, two or three recombinant proteins containing unnatural amino acids are expressed in animal cells; each recombinant protein contains The unnatural amino acids are the same or different.
  • any one of the aforementioned methods of the present invention for expressing recombinant proteins containing unnatural amino acids in animal cells is characterized in that (2) the step of transforming animal cells further includes mutating eRF1 to weaken the binding of eRF1 to mRNA and/or ribosomes .
  • the present invention provides a mutant eRF1, which is characterized in that compared with wild-type eRF1, the mutant eRF1 has a lower binding ability to mRNA and/or ribosomes, and the mutant eRF1 includes Mutations at E55, Y125, N129.
  • the mutated eRF1 of the present invention is characterized in that the mutated eRF1 includes mutations at one, two, three or four sites selected from E55R, E55D, Y125F, and N129P.
  • the application of the mutated eRF1 of the present invention in improving the read-through efficiency of the termination codon is characterized in that the termination codon is a premature termination codon (PTC, Premature termination codon).
  • PTC premature termination codon
  • the application of the mutated eRF1 in the present invention to improve the efficiency of stop codon read-through is characterized in that the mutated eRF1 and the mRNA containing PTC are expressed in the same cell.
  • the application of the mutated eRF1 of the present invention in improving the efficiency of stop codon read-through is characterized in that the PTC-containing mRNA encodes a protein containing unnatural amino acids, and the cell is a recombinant mammalian cell.
  • the application of the mutated eRF1 of the present invention in improving the efficiency of stop codon read-through is characterized in that the cell also expresses the aminoacyl-tRNA synthetase in any one of the aforementioned gene codon expansion compositions of the present invention and mutant tRNAs.
  • the present invention provides any one of the aforementioned gene codon expansion compositions, and/or the application of any one of the aforementioned mutated eRF1 in the recombinant expression of recombinant proteins containing unnatural amino acids.
  • the application of the present invention is characterized in that the recombinant expression uses mammalian cells as hosts,
  • the recombinant protein containing unnatural amino acid includes:
  • Two or three recombinant proteins each containing at least one unnatural amino acid.
  • the invention first realizes the expansion of four different systems of Mbpyl, Mmpyl, Ecleu, and EcTyr and the recoding of unnatural amino acids on three different stop codons, accurately verifies the orthogonality between different systems, and screens out the read-through rate Three systems with high orthogonality were combined Mmpyl-tRNA pyl UCA , EcTyr-tRNA Tyr UUA , Ecleu-tRNA leu CUA .
  • the anticodon loops of tRNA Pyl and tRNA Leu were not bound by their synthetases (Fig. 1B,C). It shows that the anticodon loop base mutations on tRNA Pyl and tRNA Leu will not affect the overall interaction between them and the corresponding synthetases.
  • the anticodon loop of tRNA Tyr forms hydrogen bonds with D286 and C231 in TyrRS (Fig. 1E-G), successful aminoacylation.
  • co-expression plasmids of Escherichia coli tyrosyl-tRNA synthetase (OMeYRS) and tRNA EcTyr .
  • the co-expression plasmids include the OMeYRS expression cassette controlled by the CMV promoter, the tRNA EcTyr expression cassette controlled by the U6 promoter, the tRNA EcTyr expression cassette controlled by the H1 promoter, and neomycin, kanamycin, and ampicillin resistance screening Marking; where the anticodon region of tRNA EcTyr was mutated to CUA, UCA or UUA (Fig. 2A).
  • Figure 4 Read-through of three fluorescent proteins incorporating different PTC stop codons in a cell
  • Glu55 is involved in the recognition of stop codons by directly interacting with adenine at the second position of UAA. It also forms hydrogen bonds with A3732 on the 28S rRNA and Y125 on eRF1 itself.
  • Glu55 is mutated to arginine, the positively charged guanidine group moves to the opposite side, giving up the interaction with A5 (stop codon) and A3732 (28S rRNA).
  • Asp55 and A5 is much weaker.
  • Enriched GFP+ proteins were purified with His-tag-Ni-NTA resin and diluted in PBS buffer after solution exchange. Coomassie brilliant blue staining and western blot analysis showed that the molecular weight of mutant GFP containing 3 UAAs was similar to that of wild-type GFP, which also indicated that there was no misbinding of other UAAs and endogenous AAs (Fig. 6A,B).
  • the electrophoretic bands were sheared and subjected to whole protein profiling (Fig. 6C) and peptide mass spectrometry (Fig. 6D-F), results showing successful incorporation of triple UAAs at expected sites without misincorporation. These results demonstrate that three different UAAs are successfully integrated into a single protein at specific sites in mammalian cells, facilitated by four eRF1 mutants. Successful integration of up to three UAAs into one protein will provide a powerful tool for studying protein structure and function.
  • tRNA Mmpyl tRNA Mmpyl
  • MmPylRS pyrrolysyl-tRNA synthetase
  • tRNA Anap leucine synthase
  • tRNA Anap tRNA Anap
  • Escherichia coli Escherichia coli tyrosine synthetase
  • OMeYRS Escherichia coli tyrosine synthetase
  • tRNA Tyr tRNA
  • tRNA Mbpyl tRNA Mbpyl
  • MbPylRS pyrrolysyl-tRNA synthetase
  • Example 1 Four unnatural amino acid systems expanded into 12 different systems
  • tRNA MbPyl and tRNA EcTyr were expressed by the H1 and U6 promoters
  • tRNA Ecleu was expressed by the H1 promoter
  • tRNA MmPyl was expressed by the 7sk promoter.
  • the anticodons of the tRNAs of the four nonsense systems were mutated to UCA, CUA or UUA by a point mutation step (see Figure 2A).
  • the primer sequences required for the establishment of the vectors of four different gene codon extension systems are shown in Table 1, and the primers used for the anticodon mutation of the tRNA of the four nonsense systems are shown in Table 2.
  • tRNA2 UUA MbPyl -R AATAGATCACACGGACTTTAAATCCGTGCAGGCGG tRNACUA EcLeu -F GCGAACGCCGAGGGATTTGAAATCCCTTGTGTCTACCG tRNACUA EcLeu -R CGGTAGACACAAGGGATTTCAAAATCCCTCGGCGTTCGC tRNAUUA EcLeu -F GAACGCCGAGGGATTTAAAATCCCTTGTGTCTACC tRNA UUA EcLeu -R GGTAGACACAAGGGATTTTAAATCCCTCGGCGTTC tRNA1UCA Tyr -F GTCTGTGACGGCAGATTTGAAGTCTGCTCCCTTTGGCC tRNA1 UCA Tyr -R GGCCAAAGGGAGCAGACTTCAAATCTGCCGTCACAGAC tRNA1 UUA Tyr -F CTGTGACGGCAGATTTAAAGTCTGCTCCCTTTGGC tRNA1 UUA Tyr -R GCCAAAGGGAGCAGACTTC
  • the transfection reagent MegaTran 1.0 (Origene) was used for transfection.
  • HEK293T cells were seeded in 6-well plates (Corning) until 60-70% confluent, and then transfected with 3 ⁇ g of system plasmid and mutated GFP plasmid.
  • the medium was replaced with fresh DMEM or DMEM containing 1mM UAAs (NAEK, eBK, and pAcF) or 10 ⁇ M pAnap, cultured for 48 hours, and the fluorescence intensity of the cells was observed under a fluorescent microscope and a confocal microscope (Nikon), See Figure 2B for results.
  • HEK293T was co-transfected with tRNA Mmyl UCA/CUA/UUA , tRNA Mbyl UCA/CUA/UUA , tRNA Ecleu UCA/CUA/UUA and tRNA EcTyr UCA/CUA/UUA with three different GFP nonsense mutants and related synthetases cells, observe the fluorescence of GFP, and perform flow cytometry analysis to compare the fluorescence intensity.
  • HEK293T cells 48 hours after transfection with the mutant GFP reporter gene, HEK293T cells were dissociated into single cells using trypsin/EDTA and analyzed on a BD FACSAriaTM (BD Biosciences) with appropriate filter settings (488 nm coherent sapphire laser excitation of GFP) . Front and side scatter were used to identify intact cells, and the average background fluorescence of untransfected cells was subtracted from the measured signal. Data were analyzed by FlowJo software (FlowJo). The result is shown in Figure 3D
  • GFP39TAG -R AAGTTTTCCCTATGTTGCATC GFP39TGA -F GATGCAACATGAGGAAAACTT
  • GFP39TGA -R AAGTTTTCCTCATGTTGCATC GFP 39TAA -F GATGCAACATAAGGAAAACTT
  • the system plasmid contains Mmpyl-tRNA pyl UCA , EcTyr-tRNA Tyr UUA , Ecleu-tRNA leu CUA , and they were transfected together into HEK293T cells, and 8 groups of experiments were designed, adding 0 species, 1 species, 2 species, Three different unnatural amino acids, after 48 hours, the cells were collected, the protein was extracted for Western blotting, and the expression of the three fluorescent proteins was detected under different experimental conditions. The specific results are shown in Figure 4.
  • GFP39TAG -R AAGTTTTCCCTATGTTGCATC GFP 101TGA -F ATATTTTTCTGAGATATTCGACGGG
  • GFP was mutated to GFP 39TAG-101TGA-172TAA by point mutation method, and one, two, three eRF1 mutants and plasmids of three systems, GFP plasmids containing three nonsense mutation sites were simultaneously transfected into 293T cells, and added three unnatural amino acids. After 48 hours, GFP fluorescence was observed and analyzed by flow cytometry, and the difference in GFP read-through rate under different mutant conditions was compared. The specific results are shown in Table 9.
  • HEK293T cells transfected with the three systems and all four eRF1 mutants were collected and resuspended in RIPA lysate for 30 min on ice.
  • the resulting cell lysate was subsequently clarified at 22000 xg for 10 minutes.
  • the supernatant was removed and His-tag-GFP was purified on His-tag-Ni-NTA resin (Abbkine) following the manufacturer's protocol.
  • purified GFP protein was obtained by ultrafiltration, followed by dilution in PBS buffer. Protein concentrations were determined using the BCA protein quantification kit (Thermo-Scientific).
  • HEK293T eRF1-E55R, eRF1-E55D, eRF1-Y125F, and eRF1-N129P cells were co-transfected with three UAA system plasmids and three nonsense mutant GFPs. Two days later, total protein was extracted from 8.7 ⁇ 108 cells, and then GFP protein was purified by SDS-PAGE and Commassie brilliant blue staining. GFP and adjacent bands were excised and sent for MS analysis (Thermo-LUMOS). GFP was digested with trypsin at residues 39 and 101, and with Asp-N protease (V162, promega) at residue 172. The molecular mass of purified GFP was analyzed by SYNAPT-G2-Si high resolution ion mobility mass spectrometer (Waters). The specific results are shown in Figure 6.
  • sequence of the main nucleic acid element of the present invention is as follows:

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Abstract

一种在动物细胞中表达含有非天然氨基酸重组蛋白的方法，在四组氨基酰-tRNA合成酶和相应tRNA的基础上，构建识别终止密码子的氨基酰-tRNA，筛选获得三种基因密码子拓展系统。所述三种基因密码子拓展系统对终止密码子通读效率高、相互兼容性强，从而可以在一条外源蛋白上插入三种非天然氨基酸、或对同一细胞的三条外源蛋白上分别插入不同的非天然氨基酸。为进一步提高通读率，还对释放因子eRF1进行了突变以减弱其与终止密码子的相互作用。

Description

一种含非天然氨基酸蛋白的制备方法 技术领域
本发明属于生物制药领域，具体涉及一种含非天然氨基酸蛋白的制备方法，尤其涉及一种在哺乳动物细胞中实现一个蛋白同时高效定点插入三种不同非天然氨基酸的方法。
背景技术
自然界中总共有64个三联体密码子，在一般的生物体中，其中61个密码子编码20种天然氨基酸，而另外三种密码子(UAA,UGA,UAG)不编码任何氨基酸，当核糖体翻译到这些密码子的时候，会有正常的终止因子来终止蛋白质翻译。随着更多的生物学现象被揭示，科学家们发现在古细菌中，终止密码子之一的琥珀终止密码子UAG被用来编码特殊的氨基酸到必须的蛋白质中，以保证细菌正常的生长。基于此，科学家们将古细菌中的tRNA和tRNA合成酶对单独拿出来，经过正负筛选的优化，使其在大肠杆菌中利用本来不编码氨基酸的UAG密码子，来编码自然界中不存在的氨基酸(即非天然氨基酸)到蛋白质的特定位置。
基于不同体系的非天然氨基酸已经被发现并应用于研究工作中，包括酪氨酸体系，吡咯赖氨酸体系，苯丙氨酸体系，大大丰富了非天然氨基酸结构的选择；而越来越多的物种，包括大肠杆菌，哺乳动物细胞，酵母，昆虫细胞等，都可以进行非天然氨基酸的插入，也为这套技术的广泛应用奠定了基础；在插入方法上，从最早的琥珀终止密码子，到其他终止密码子，四联体密码子，稀有密码子，甚至优化的特殊核糖体等，为插入方法提供了更多的选择。
基因密码子扩展技术从开发至今，已经在分子探针、药物靶标发现、药物改良修饰以及各种生化领域上得到了很广泛的应用。通过引入不同的非天然氨基酸，科学家们可以选择性的赋予蛋白质更新型的性质，以发现更多的生命科学中的奥秘。尽管如此，基因密码子扩展技术掺入氨基酸仍然存在效率低下、易错配等技术问题。在氨基酸掺入中，有可能会出现三种类型的错配：(1)tRNA/aaRS；(2)aaRS/aminoacid；and(3)tRNA/codon。体内将氨基酸类似物掺入蛋白质的一种方法就是借助于氨酰tRNA合成酶的疏漏，催化某种氨基酸类似物连接到tRNA从而进入蛋白质。在非天然氨基酸掺入中，能够避免所有三种错配的tRNA/aaRS需要设计相应的正交系统，仅仅在有其相应密码子的正确的位点插入新的氨基酸。也就是说，要扩展遗传密码，将新的氨基酸引入生物合成体系，需要有识别正交密码子的tRNA、对应的能将非天然氨基酸装上tRNA的高特异性aaRS，从而实现非天然氨基酸的掺入。正交系统需满足：唯一的新密码子，能识别新密码子的tRNA，能与其偶联的aaRS；并且，aaRs/tRNA组合必须与内源的aaRS/tRNA组合是正交的(即不可交叉使用)，以保证非天然氨基酸掺入的精度。
包括抗体在内的蛋白质药物是国际上发展最快、利润最高、竞争也最激烈的研发领域,然而几乎所有蛋白质药物都存在以下共性的重大缺陷：①在体内易被蛋白酶降解致半衰期过短；②分子量大免疫源性强,机体注射诱导体内产生中和抗体致药效不断降低；③不能定点修饰,缺乏像小分子药物进行构效关系的系统研究手段等。这些瓶颈困扰着蛋白质药物，特别是质量控制和药效方面的整体发展。
在蛋白质药物的改良上，通过将含有苯丙酮的非天然氨基酸引入到蛋白质的特定位点，并基于苯丙酮和羟乙胺的化学偶联反应，美国的Ambrx公司已开发了一系列蛋白质药物新产品，包括双功能抗体，抗体药物偶联物等。除此外，借助琥珀终止密码子(UAG)和DNA定点突变技术，在干扰素或者其他蛋白药物的多个潜在的可修饰位置 上引入了含叠氮基团的非天然氨基酸作为修饰“把手”，进而通过Click反应与含DIBO基团的不同分子量的PEG分子进行偶联。同时对引入非天然氨基酸的条件以及PEG的偶联条件进行了优化，成功建立了蛋白质药物温和、快速、高效、定量和特异的修饰方法。
绿色荧光蛋白GFP是最常用的报告基因，也是指示非天然氨基酸插入的有力工具，其由238个氨基酸组成。本申请以绿色荧光蛋白GFP为例说明本发明的技术背景，但是下述内容无论如何都不能理解为其是现有技术的承认，也无论无何都不能认为本发明的仅仅适用于GFP蛋白。
现在的蛋白药物的大量表达及修饰仍主要在细菌中进行，在哺乳动物细胞中非天然氨基酸插入效率低，特别是当表达多个非天然氨基酸修饰位点的多肽时，不同的密码子扩展系统之间还会相互干扰，不仅影响通读效率、而且也导致修饰位点氨基酸残基的不确定性。
目前现有技术中基因水平上非天然氨基酸蛋白质修饰仅能以单个位点、一种非天然氨基酸修饰，无法实现一个蛋白的多个位点特异性修饰、多个蛋白不同非天然氨基酸修饰，这些问题成为了基因密码子扩展技术在蛋白质药物开发中应用的瓶颈。因此，如何将外源的基因密码子系统应用于哺乳动物细胞，提高哺乳动物细胞中非天然氨基酸残基多肽的修饰效率，实现在一个蛋白多个位点同时特异性修饰、或多种带有不同非天然氨基酸残基蛋白的同时表达是本领域亟待解决的技术问题。
发明内容
针对上述现有技术中存在的缺陷，本发明的目的在于提供一种在哺乳动物细胞中实现一个蛋白同时高效定点插入三种不同非天然氨基酸的方法。本发明提供了一种在动物细胞中表达含有非天然氨基酸重组蛋白的方法，在四组氨基酰-tRNA合成酶和相应tRNA的基础上，构建识别终止密码子的氨基酰-tRNA，筛选获得三种基因密码子拓展系统。所述三种基因密码子拓展系统对终止密码子通读效率高、相互兼容性强，从而可以在一条外源蛋白上插入三种非天然氨基酸、或对同一细胞的三条外源蛋白上分别插入不同的非天然氨基酸。为进一步提高通读率，还对释放因子eRF1进行了突变以减弱其与终止密码子的相互作用。
本发明所采用的的技术方案如下：
基于四种不同基因密码子拓展系统酶和tRNA识别的结构特点，拓展构建12种不同系统的质粒载体。基于Mbpyl，Mmpyl,Ecleu，EcTyr四种基因密码子拓展系统的酶和tRNA的识别不影响非天然氨基酸编码的特点，将四种系统的tRNA反密码子环突变为CUA,UCA,UUA,通过点突变和分子克隆方法构建含有正交的氨酰tRNA合成酶和对应的串联tRNA的十二种不同基因密码子拓展系统的高效真核表达载体，并且构建在39位突变为UAA,UAG,UGA的GFP报告基因表达载体。通过GFP荧光流式分析方法，筛选出三种高通读率且正交性好的基因密码子拓展系统。分别在aaRS/tRNA、aaRS/UAA、tRNA/stop codon层次上，将12种不同系统进行实验组合和GFP蛋白荧光流式分析，精确筛选高通读效率且正交的基因密码子拓展系统Mmpyl-tRNA pyl UCA,EcTyr-tRNA Tyr UUA,Ecleu-tRNA leu CUA。基于此，完成了在一个哺乳动物细胞中同时完成三种正交系统分别插入三种不同荧光蛋白RFP,BFP,GFP,实现三种蛋白的定点修饰，为一个蛋白上多点修饰奠定了基础。为同时提高一个蛋白上三种终止密码子的通读效率，通过GFP蛋白流式分析系统精确评价eRF1不同位点突变体单个或者多个组合对三种不同来源的正交的基因密码子拓展系统的通读效率影响，确定最佳同时提高三系统(三种终止密码子)通读率的eRF1突变体组合(E55R,E55D,N129P,Y125F)。 转染eRF1突变体组合进入细胞后，对三位点定点修饰的GFP蛋白进行扩增表达，并进行蛋白纯化和LC-MS质谱等一系列验证，获得高纯度的多位点特异性修饰蛋白。
具体而言：
一方面，本发明提供一种基因密码子拓展组合物，包括：
(1)一种或多种氨基酰-tRNA合成酶，结合突变的tRNA；
(2)一种或多种突变的tRNA，所述突变的tRNA反密码子环上突变为终止密码子的互补序列；
其中，氨基酰-tRNA合成酶能将突变的tRNA结合到非天然氨基酸上产生氨基酰-tRNA。
进一步，本发明所述基因密码子拓展组合物，其特征在于：
所述氨基酰-tRNA合成酶选自Pyl-tRNA合成酶(PylRS)、Leu-tRNA合成酶(LeuRS)、Tyr-tRNA合成酶(TyrRS)；
所述突变的tRNA选自tRNA Pyl、tRNA Leu、tRNA Tyr。
进一步，本发明所述基因密码子拓展组合物，其特征在于tRNAPyl和PylRS来自马氏甲烷八叠球菌(Methanosarcina mazei)或巴氏甲烷八叠球菌(Methanosarcina barkeri)；tRNALeu和LeuRS来自大肠杆菌；tRNATyr和TyrRS来自大肠杆菌。
进一步，本发明所述基因密码子拓展组合物，其包括选自以下氨基酰-tRNA合成酶与突变tRNA组合中的一种或多种
MmPylRs和tRNA MmPyl UCA、
MmPylRs和tRNA MmPyl CUA、
MmPylRs和tRNA MmPyl UUA、
MbPylRs和tRNA MbPyl UCA、
MbPylRs和tRNA MbPyl CUA、
MbPylRs和tRNA MbPyl UUA、
EcLeuRs和tRNA EcLeu UCA、
EcLeuRs和tRNA EcLeu CUA、
EcLeuRs和tRNA EcLeu UUA、
OMeYRs和tRNA EcTyr UCA、
OMeYRs和tRNA EcTyr CUA、
OMeYRs和tRNA EcTyr UUA。
进一步，本发明所述基因密码子拓展组合物，其包括
选自MmPylRs和tRNA MmPyl UCA、MmPylRs和tRNA MmPyl CUA、MmPylRs和tRNA MmPyl UUA、MbPylRs和tRNA MbPyl UCA、MbPylRs和tRNA MbPyl CUA、MbPylRs和tRNA MbPyl UUA中任一种的氨基酰-tRNA合成酶与突变tRNA组合；
选自EcLeuRs和tRNA EcLeu UCA、EcLeuRs和tRNA EcLeu CUA、EcLeuRs和tRNA EcLeu UUA中任一种的氨基酰-tRNA合成酶与突变tRNA组合；
以及选自OMeYRs和tRNA EcTyr UCA、OMeYRs和tRNA EcTyr CUA、OMeYRs和tRNA EcTyr UUA中任一种的氨基酰-tRNA合成酶与突变tRNA组合。
进一步，本发明所述基因密码子拓展组合物，其包括MmPylRs和tRNA MmPyl UCA、OMeYRs和tRNA EcTyr UUA、以及EcLeuRs和tRNA EcLeu CUA三组氨基酰-tRNA合成酶与突变tRNA的组合。
进一步，本发明前述任一项基因密码子拓展组合物，其特征在于还包括非天然氨基酸，所述非天然氨基酸优选NAEK、Anap、pAcF。
第二方面，本发明提供一种在动物细胞中表达含有非天然氨基酸重组蛋白的方法，包括：
(1)制备重组蛋白表达盒，其中重组蛋白编码序列中非天然氨基酸的密码子设置为终止密码子；
(2)改造动物细胞，使其表达权利要求1-7中任一项基因密码子拓展组合物中的氨基酰-tRNA合成酶和突变的tRNA；
(3)将步骤(1)中重组蛋白表达盒导入步骤(2)改造的动物细胞，制备重组动物细胞；
(4)将步骤(3)构建的重组动物细胞在补充添加非天然氨基酸的培养基中培养，诱导含有非天然氨基酸重组蛋白的表达。
进一步，本发明所述在动物细胞中表达含有非天然氨基酸重组蛋白的方法，其特征在于所述重组蛋白氨基酸序列中含有一种、两种或三种非天然氨基酸。
进一步，本发明所述在动物细胞中表达含有非天然氨基酸重组蛋白的方法，其特征在于在动物细胞中表达一种、两种或三种含有非天然氨基酸的重组蛋白；各重组蛋白中含有的非天然氨基酸种类相同或不同。
进一步，本发明前述任一项在动物细胞中表达含有非天然氨基酸重组蛋白的方法，其特征在于(2)改造动物细胞的步骤还包括突变eRF1以削弱eRF1与mRNA、和/或核糖体的结合。
第三方面，本发明提供一种突变的eRF1，其特征在于与野生型eRF1相比，所述突变的eRF1对mRNA、和/或核糖体具有较低的结合能力，并且所述突变的eRF1包括E55、Y125、N129位点的突变。
进一步，本发明所述突变的eRF1，其特征在于所述突变的eRF1包括选自E55R、E55D、Y125F、N129P中的一个、两个、三个或四个位点的突变。
进一步，本发明前述任一项突变的eRF1在提高终止密码子通读效率中的应用。
进一步本发明所述突变的eRF1在提高终止密码子通读效率中的应用，其特征在于所述终止密码子为提前终止密码子(PTC，Premature termination codon)。
进一步，本发明所述突变的eRF1在提高终止密码子通读效率中的应用，其特征在于使所述突变的eRF1和含有PTC的mRNA在同一细胞内表达。
进一步，本发明所述突变的eRF1在提高终止密码子通读效率中的应用，其特征在于所述含有PTC的mRNA编码含有非天然氨基酸的蛋白，所述细胞为重组哺乳动物细胞。
进一步，本发明所述突变的eRF1在提高终止密码子通读效率中的应用，其特征在于所述细胞中还表达有本发明前述任一项基因密码子拓展组合物中的氨基酰-tRNA合成酶和突变的tRNA。
第四方面，本发明提供前述任一项基因密码子拓展组合物、和/或前述任一项所述突变的eRF1在重组表达含非天然氨基酸重组蛋白中的应用。
进一步，本发明所述的应用，其特征在于所述重组表达以哺乳动物细胞为宿主，
所述含非天然氨基酸重组蛋白包括：
含有一个、两个或三个非天然氨基酸的一种重组蛋白，或者
分别含有至少一个非天然氨基酸的两种或三种重组蛋白。
本发明首先实现了Mbpyl，Mmpyl,Ecleu，EcTyr四种不同系统的拓展和非天然氨基酸在三种不同终止密码子上的重编码，精确验证不同系统之间的正交性，并筛选出通读率高正交性好的三种系统组合Mmpyl-tRNA pyl UCA,EcTyr-tRNA Tyr UUA,Ecleu-tRNA leu CUA。在此基础上，首次系统精确评价了eRF1不同位点突变体单个或者多个组合对三种不同来源的基因密码子拓展系统的通读效率影响，筛选出最佳的四种eRF1突变体组合(E55R,E55D,Y125F,N129P)，条件性减弱释放因子eRF1与三种不同终止密码子之间的相互作用力，从而整体性地提高了三种不同正交系统的通读效率，从0.2％提高到12％， 实现了三种不同非天然氨基酸在哺乳动物细胞中同时高效定点插入。并优化了表达纯化和蛋白鉴定方法，得到高产量高纯度的蛋白。该方法可适用于哺乳动物细胞中其他的蛋白多位点特异性修饰。
附图说明
通过参考附图阅读下文的详细描述，本公开示例性实施方式的上述以及其他目的、特征和优点将变得易于理解。在附图中，以示例性而非限制性的方式示出了本公开的若干实施方式，并且相同或对应的标号表示相同或对应的部分，其中：
图1四种基因密码子拓展系统氨酰tRNA合成酶和tRNA相互作用
四种系统是通过它们的突变反密码子来识别提前终止密码子。为了研究反密码子突变是否会影响这些tRNA和相应合成酶之间的整体相互作用，我们研究了合成酶和tRNA的三维结构对接。我们使用了除pylMmRS(PDB:2ZIM)之外的hafniense脱硫杆菌的可用复合物，这两种合成酶在结构上是保守的(图1A)。
根据合成酶和相应的tRNA的结合结构，tRNA Pyl和tRNA Leu的反密码子环不受其合成酶的结合(图1B，C)。表明tRNA Pyl和tRNA Leu上的反密码子环碱基突变不会影响其和相应的合成酶之间的整体相互作用。
tRNA Tyr的反密码子环与TyrRS中的D286和C231形成氢键(图1D)，但突变的反密码子UUA、CUA和UCA与TyrRS中的上述关键残基仍能保持弱的相互作用(图1E-G)，成功氨基酰化。
图1的结构分析表明，上述四种系统使用三种突变tRNA均可以完成氨基酰化过程，可编码非天然氨基酸，进而实现密码子系统的拓展。
图2四种单密码子拓展系统质粒构建及含PTC位点GFP的通读
构建大肠杆菌酪氨酰-tRNA合成酶(OMeYRS)和tRNA EcTyr共表达质粒。所述共表达质粒包括CMV启动子控制的OMeYRS表达盒、U6启动子控制的tRNA EcTyr表达盒、H1启动子控制的tRNA EcTyr表达盒、以及新霉素、卡那霉素、氨苄青霉素抗性筛选标记；其中tRNA EcTyr的反密码子区突变为CUA、UCA或UUA(图2A)。
利用转入OMeYRS-tRNA EcTyr共表达质粒的宿主细胞过表达插入三种终止密码子的重组GFP时，如培养基中不加入pAcF，不产生荧光；加入pAcF产生荧光。上述结果表明，不加入非天然氨基酸的情况下，重组GFP基因在插入的终止密码子处结束翻译；加入非天然氨基酸pAcF，OMeYRS-tRNA EcTyr系统能够在重组GFP基因插入的终止密码子处加上pAcF实现通读，产生荧光。tRNA EcTyr上使用不同的反密码子通读效率不同，CUA>UAA>UCA(图2B)。
其他三种系统拓展的质粒构建原理和GFP通读验证与OMeYRS-tRNA EcTyr系统一致。不加入相应非天然氨基酸时，均不产生荧光，加入相应非天然氨基酸条件下，均可实现成功通读。图2结果表明成功构建了12种不同的基因密码子拓展系统。
图3不同密码子拓展系统之间的正交性分析
为了排除不同系统的潜在相互作用，我们验证了四种可用aaRS/tRNA系统的相互正交性(图3C)。
首先，我们在UAA(非天然氨基酸，unnatural amino acid)和aaRS/tRNA对水平上进行了正交性分析，结果如图3A。结果表明，Anap和pAcF表现出很大的正交性，NAEK与MbpylRS-tRNA Mbpyl表现出轻微的交叉反应性。但在加入eBK后，MmpylRS-tRNA Mmpyl和AnapRS-tRNA EcLeu均可读取终止密码子，表明eBK不适合用于三个提前终止密码子同时编码非天然氨
基酸实验中。
进一步，我们在合成酶和tRNAs水平上进行正交性分析，结果如图3B。将合成酶分别共转染到具有不同tRNA CUA的HEK293T细胞中，以及含有TAG突变的GFP，结果表明，只有合成酶与相对应系统的tRNA CUA组别，GFP可以产生荧光。
随后，我们用三种不同的无义抑制tRNA评估了非同源终止密码子的错配潜力。将tRNA Mmyl UCA/CUA/UUA、tRNA Mbyl UCA/CUA/UUA、tRNA Ecleu UCA/CUA/UUA和tRNA EcTyr UCA/CUA/UUA与三种不同的GFP(39TGA/TAG/TAA)无义突变体以及相关合成酶共转染HEK293T细胞，结果如图3D。在没有任何错配的情况下，只有当tRNA质粒与含有同源无义密码子的报告者结合时，GFP才会发出荧光。这些结果进一步证实，MmPyl、EcTyr和EcLeu三系统的组合适用于哺乳动物细胞三种提前终止密码子的高保真共通读。
图4一个细胞中三种掺入不同PTC终止密码子荧光蛋白的通读
我们验证了三个系统同时将三种UAA同时插入到三种不同蛋白质中的可行性。我们构建了RFP 36TGA、BFP 39TAG和GFP 39TAA突变体，并将它们一起转染到HEK293T细胞中，质粒含Mmpyl-tRNA pyl UCA,EcTyr-tRNA Tyr UUA，Ecleu-tRNA leu CUA。根据Western bloting结果，我们发现这些蛋白只有在加入相应的UAA后才会表达；当加入两种UAA时，只有两种相关蛋白表达；当添加三种UAA时，所有三种荧光蛋白将同时表达。结果表明，三种系统可以独立工作，同时将UAAs成功插入单个哺乳动物细胞中的三种不同蛋白质中，具体结果见图4。
图5 eRF1四种突变体与终止密码子相互作用的结构分析
我们试图在更深层次上研究eRF1突变对提高系统通读的作用。Glu55是通过直接与UAA第二位置的腺嘌呤相互作用参与终止密码子的识别。它还与28S rRNA上的A3732和eRF1本身上的Y125形成氢键。当Glu55突变为精氨酸时，带正电的胍基会向相反的一侧移动，放弃与A5(终止密码子)和A3732(28S rRNA)的相互作用。即使突变为天冬氨酸，Asp55和A5之间的相互作用也会弱得多。虽然Y125F和N129P突变对Glu55与A5和A3732的相互作用影响不大，但它们可能通过改变YxCxxxF基序的方向和进一步削弱eRF1与mRNA之间的结合间接起作用。总体而言，E55R,E55D,Y125F,N129P四个氨基酸位点突变可明显减弱eRF1与终止密码子的识别结合，从而提高哺乳动物细胞中提前终止密码子的通读率，具体结果见图5。
图6蛋白表达纯化及质谱鉴定结果
用His-tag-Ni-NTA树脂纯化富集的GFP+蛋白，并在溶液交换后在PBS缓冲液中稀释。考马斯亮蓝染色和western blot分析表明，含有3个UAA的突变GFP的分子量与野生型GFP的分子量相似，也表明没有其他UAA和内源性AA错误结合(图6A，B)。剪切电泳条带，并进行全蛋白质谱分析(图6C)和肽质谱分析(图6D-F)，结果表明在预期位点成功地将三重UAAs掺入，而没有错误掺入。这些结果表明，在四个eRF1突变体的促进下，三种不同的UAA在哺乳动物细胞的特定位点成功地整合到单个蛋白质中。成功地将多达三种UAA整合到一种蛋白质中，将为研究蛋白质结构和功能提供一个强有力的工具。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
利用来自马氏古甲烷球菌(Methanosarcina mazei)的tRNA(tRNA Mmpyl)和吡咯赖氨酰-tRNA合成酶(MmPylRS)，来自大肠杆菌的亮氨酸合成酶(AnapRS)和tRNA(tRNA Anap)，来自大肠杆菌酪氨酸合成酶(OMeYRS)和tRNA(tRNA Tyr)，来自巴氏古甲烷球菌(Methanosarcina barkeri)的tRNA(tRNA Mbpyl)和吡咯赖氨酰-tRNA合成酶(MbPylRS)，以绿色荧光蛋白GFP为模式，说明本发明的技术方案。
实施例1 四种非天然氨基酸系统拓展成12种不同的系统
我们使用了除pylMmRS(PDB:2ZIM)之外的hafniense脱硫杆菌的可用复合物，这两种合成酶在结构上是保守的。根据合成酶和相应的tRNA的结合结构，tRNA Pyl和tRNA Leu的反密码子环不受其合成酶的结合。tRNA Tyr的反密码子环确实与TyrRS中的D286和C231形成氢键，但突变的反密码子UUA、CUA和UCA与TyrRS中的上述关键残基保持弱相互作用，从而确保氨基酰化成功(参见图1)。
为了构建编码四种氨酰tRNA合成酶(OMeYRS、EcleuRS、MbPylRS和MmpylRS)的质粒，通过双酶切反应将在CMV启动子下表达的相应基因分别插入到pcDNA3.1(+)载体中。tRNA MbPyl和tRNA EcTyr由H1和U6启动子表达，tRNA Ecleu由H1启动子表达，tRNA MmPyl由7sk启动子表达。为了创建tRNA质粒，我们通过相应的酶切方法将4个或者8个tRNA基因拷贝分别整合到pcDNA3.1(+)载体中。四个无义系统的tRNA的反密码子通过点突变步骤突变为UCA、CUA或UUA(参见图2A)。四种不同基因密码子拓展系统载体建立所需的引物序列如表1所示，四个无义系统的tRNA的反密码子突变所用引物如表2所示。
表1.四种不同基因密码子拓展系统载体建立所需的引物序列
Figure PCTCN2022117667-appb-000001
表2.四种不同基因密码子拓展系统拓展tRNA突变所用的引物序列
tRNA Sequence
tRNA UCA MmPyl-F AACCCGGCTGAACGGATTTGAAGTCCATTCGATCTACATG
tRNA UCA MmPyl-R CATGTAGATCGAATGGACTTCAAATCCGTTCAGCCGGGTT
tRNA UUA MmPyl-F CCGGCTGAACGGATTTAAAGTCCATTCGATCTACA
tRNA UUA MmPyl-R TGTAGATCGAATGGACTTTAAATCCGTTCAGCCGG
tRNA1 UCA MbPyl-F AACCCGGCTGAACGGATTTGAAGTCCGTTCGATCTACATG
tRNA1 UCA MbPyl-R CATGTAGATCGAACGGACTTCAAATCCGTTCAGCCGGGTT
tRNA1 UUA MbPyl-F CCGGCTGAACGGATTTAAAGTCCGTTCGATCTACA
tRNA1 UUA MbPyl-R TGTAGATCGAACGGACTTTAAATCCGTTCAGCCGG
tRNA2 UCA MbPyl-F ACCCGCCTGCACGGATTTGAAGTCCGTGTGATCTATTC
tRNA2 UCA MbPyl-R GAATAGATCACACGGACTTCAAATCCGTGCAGGCGGGT
tRNA2 UUA MbPyl-F CCGCCTGCACGGATTTAAAGTCCGTGTGATCTATT
tRNA2 UUA MbPyl-R AATAGATCACACGGACTTTAAATCCGTGCAGGCGG
tRNA CUA EcLeu-F GCGAACGCCGAGGGATTTGAAATCCCTTGTGTCTACCG
tRNA CUA EcLeu-R CGGTAGACACAAGGGATTTCAAATCCCTCGGCGTTCGC
tRNA UUA EcLeu-F GAACGCCGAGGGATTTAAAATCCCTTGTGTCTACC
tRNA UUA EcLeu-R GGTAGACACAAGGGATTTTAAATCCCTCGGCGTTC
tRNA1 UCA Tyr-F GTCTGTGACGGCAGATTTGAAGTCTGCTCCCTTTGGCC
tRNA1 UCA Tyr-R GGCCAAAGGGAGCAGACTTCAAATCTGCCGTCACAGAC
tRNA1 UUA Tyr-F CTGTGACGGCAGATTTAAAGTCTGCTCCCTTTGGC
tRNA1 UUA Tyr-R GCCAAAGGGAGCAGACTTTAAATCTGCCGTCACAG
tRNA2 UCA Tyr-F CCAAAGGGAGCGGATTTGAAGTCCGCCGCGTTTAGC
tRNA2 UCA Tyr-R GCTAAACGCGGCGGACTTCAAATCCGCTCCCTTTGG
tRNA2 UUA Tyr-F AAAGGGAGCGGATTTAAAGTCCGCCGCGTTTAG
tRNA2 UUA Tyr-R CTAAACGCGGCGGACTTTAAATCCGCTCCCTTT
质粒构建成功后，使用转染试剂MegaTran 1.0(Origene)进行转染。HEK293T细胞接种在6孔板(康宁)中，直至60～70％融合，然后转染3μg系统质粒和突变后的GFP质粒。转染6小时后，将培养基更换为新鲜DMEM或含有1mM UAAs(NAEK、eBK和pAcF)或10μM pAnap的DMEM,培养48小时,在荧光显微镜和共焦显微镜(尼康)下观察细胞荧光强度，结果参见图2B。
实施例2 四种非天然氨基酸系统正交性验证及筛选哺乳动物细胞中效率最高的三系统最佳组合
为了排除不同系统的相互作用，我们验证了四种可用aaRS/tRNA系统的相互正交性(图3C)。
首先，我们在UAA和aaRS/tRNA对水平上进行了正交性验证。在转染系统和相应的GFP质粒48小时后，培养基中分别加入不同的非天然氨基酸，观察GFP的荧光，并做流式分析比较荧光强度，结果如图3A所示。
然后，我们在合成酶和tRNAs水平上确定了正交性。将合成酶分别共转染到具有不同tRNA CUA的HEK293T细胞中，以及含有TAG突变的GFP，观察GFP的荧光，并做流式分析比较荧光强度，结果如图3B所示。
另外，我们用三种不同tRNA评估非同源终止密码子的错配潜力。将tRNA Mmyl UCA/CUA/UUA、tRNA Mbyl UCA/CUA/UUA、tRNA Ecleu UCA/CUA/UUA和tRNA EcTyr UCA/CUA/UUA与三种不同的GFP无义突变体以及相关合成酶共转染HEK293T细胞，观察GFP的荧光，并做流式分析比较荧光强度。在用突变GFP报告基因转染48小时后，使用胰蛋白酶/EDTA将HEK293T细胞分离成单个细胞，并在BD FACSAriaTM(BD Biosciences)上使用适当的过滤器设置(488nm相干蓝宝石激光激发GFP)进行分析。正面和侧面散射用于识别完整细胞，并从测量信号中减去未转染细胞的平均背景荧光。数据通过FlowJo软件(FlowJo)进行分析。结果如图3D所示
表3.三种不同的GFP无义突变体构建所用的引物
Primer Sequence
GFP 39TAG-F GATGCAACATAGGGAAAACTT
GFP 39TAG-R AAGTTTTCCCTATGTTGCATC
GFP 39TGA-F GATGCAACATGAGGAAAACTT
GFP 39TGA-R AAGTTTTCCTCATGTTGCATC
GFP 39TAA-F GATGCAACATAAGGAAAACTT
GFP 39TAA-R AAGTTTTCCTTATGTTGCATC
为了研究实现一个蛋白三种不同终止密码子的同时高效通读，我们比较了四个系统的十二种组合。将每个aaRS-tRNA对与相应的GFP突变体HEK293T细胞共转染，三组携带不同aaRS-tRNA对的细胞组成一个组合，并在通过His-tag-Ni-NTA树脂纯化后，使用BCA蛋白质定量试剂盒测定每个组合的GFP产量。我们比较了三个系统的不同组合的通读效率。将每个aaRS-tRNA对与相应的GFP突变体HEK293T细胞共转染，三组携带不同aaRS-tRNA对的细胞组成一个组合，转染48小时后，收集细胞进行流式分析，获得精确的GFP通读效率。GFP蛋白通过His-tag-Ni-NTA树脂纯化后，使用BCA蛋白质定量试剂盒测定每个组合的GFP实际产量，从而筛选最佳的三系统组合，结果如表4所示。通过比较GFP产量和以突变GFP产量与野生型GFP产量之比计算的通读率，我们确认，对于哺乳动物细胞中的UAA掺入，Mmpyl-tRNA pyl UCA,EcTyr-tRNA Tyr UUA，Ecleu-tRNA leu CUA的组合显示出最高的读取效率和突变GFP产量。
表4.密码子扩展系统不同组合的GFP通读率和GFP产量
Figure PCTCN2022117667-appb-000002
Figure PCTCN2022117667-appb-000003
实施例3 RFP,BFP,GFP的质粒构建及哺乳动物细胞中三荧光蛋白实现可控表达
我们首先基于点突变和分子克隆方法构建了RFP 36TGA、BFP 39TAG和GFP 39TAA突变体，构建上述3种突变荧光蛋白的引物如表5所示。
表5.三种荧光蛋白突变体构建所用的引物序列
Primer Sequence
GFP 39TAA-F GATGCAACATAAGGAAAACTT
GFP 39TAA-R AAGTTTTCCTTATGTTGCATC
RFP 36TGA-F GAGGGCGAGGGCTGACCCTACGAGGGC
RFP 36TGA-R GCCCTCGTAGGGTCAGCCCTCGCCCTC
BFP 39TAG-F GAGGGCACCTAGACCATGAGA
BFP 39TAG-R TCTCATGGTCTAGGTGCCCTC
系统质粒含Mmpyl-tRNA pyl UCA,EcTyr-tRNA Tyr UUA，Ecleu-tRNA leu CUA，将它们一起转染到HEK293T细胞中，设计8组实验，培养基中分别加入0种，1种，2种，3种不同的非天然氨基酸，48h后，收集细胞，提蛋白进行Western bloting，检测不同实验条件下，三种荧光蛋白的表达情况。具体结果见图4。
实施例4 eRF1不同突变体及不同组合对三种终止密码子的通读影响分析
以NCBI登录号NT_037436.4所示eRF1序列为基础，首先根据eRF1与终止密码子识别的结构分析(图5)，确定突变E55R,E55D,Y125F,N129P四个位点，通过点突变完成(eRF1不同突变体构建所用的引物序列如表6所示)。之后，将一个，两个，三个突变体与系统的质粒，含有一个无义突变位点的GFP(39TAG、101TGA、172TAA)质粒分别同时转染入293T细胞中，加入相应的非天然氨基酸。48h后，观察GFP荧光并进行流式分析，比较不同突变体条件下GFP通读率的差异，结果见表8。
表6.eRF1不同突变体构建所用的引物序列
Primer Sequence
eRF1-E55R-F GTGGCAAAAATGTTAGCGGATCGCTTTGGAACTGCATCTAAC
eRF1-E55R-R GTTAGATGCAGTTCCAAAGCGATCCGCTAACATTTTTGCCAC
eRF1-E55D-F GTGGCAAAAATGTTAGCGGATGATTTTGGAACTGCATCTAACAT
eRF1-E55D-R ATGTTAGATGCAGTTCCAAAATCATCCGCTAACATTTTTGCCAC
eRF1-N129P-F CGTCATTGTATTTGTGTGACCCCAAATTCCATACAGAGGCTC
eRF1-N129P-R GAGCCTCTGTATGGAATTTGGGGTCACACAAATACAATGACG
eRF1-Y125F-F CCAATTAATACGTCATTGTTCTTGTGTGACAACAAATTCCATACAGAGG
eRF1-Y125F-R CCTCTGTATGGAATTTGTTGTCACACAAGAACAATGACGTATTAATTGG
表7.突变荧光蛋白GFP(39TAG、101TGA、172TAA)构建所用引物
Primer Sequence
GFP 39TAG-F GATGCAACATAGGGAAAACTT
GFP 39TAG-R AAGTTTTCCCTATGTTGCATC
GFP 101TGA-F ATATTTTTCTGAGATATTCGACGGG
GFP 101TGA-R CCCGTCGAATATCTCAGAAAAATAT
GFP 172TAA-F CACAACATTTAAGATGGAAGC
GFP 172TAA-R GCTTCCATCTTAAATGTTGTG
表8.eRF1四种突变体组合对三种GFP(分别含不同PTC终止密码子)通读的影响
Figure PCTCN2022117667-appb-000004
表8的结果表明，eRF1四种突变体不同组合对三种不同系统的影响略有差异，其中，E55R和E55D对三种系统的影响明显高于其他的突变体。有E55R突变体的双组合，三组合的通读效率也明显高于没有该突变体的组合。表明四种突变体对三种不同系统的通读均有明显的促进作用。
实施例5 筛选同时提高一个蛋白三种提前终止密码子通读效率的最佳的eRF1不同突变体组合
首先通过点突变方法突变GFP为GFP 39TAG-101TGA-172TAA,将一个，两个，三个eRF1突变体与三个系统的质粒，含有三个无义突变位点的GFP质粒分别同时转染入293T细胞中，并加入三种非天然氨基酸。48h后，观察GFP荧光并进行流式分析，比较不同突变体条件下GFP通读率的差异。具体结果见表9。
表9.eRF1四种突变体组合对一种GFP(含三个不同PTC终止密码子)通读的影响
[援引加入(细则20.6) 09.12.2022]　
Figure WO-DOC-TABLE-9
[援引加入(细则20.6) 09.12.2022]　
表9的结果表明，与其他组合相比，四个突变体的引入明显提高了含三个不同PTC终止密码子GFP的通读效率，效率可以达到11.6%。
收集用三种系统和所有四种eRF1突变体转染的HEK293T细胞，并在冰上再悬浮在RIPA裂解液中30分钟。随后在22000×g下澄清产生的细胞裂解液10分钟。去除上清液，并按照制造商的协议在His-tag-Ni-NTA树脂(Abbkine)上纯化His-tag-GFP。然后，通过超滤获得纯化的GFP蛋白，然后在PBS缓冲液中稀释。使用BCA蛋白质定量试剂盒(Thermo-Scientific)测定蛋白质浓度。此外，将三种UAA系统的质粒和三种无义突变的GFP共转染HEK293T(eRF1-E55R、eRF1-E55D、eRF1-Y125F和eRF1-N129P)细胞。两天后，从8.7×10 8细胞中提取总蛋白，然后通过SDS-PAGE和Commassie亮蓝染色纯化GFP蛋白。切下GFP和附近的条带并发送至MS分析(Thermo-LUMOS)。GFP上的残基39和101位用胰蛋白酶消化，残基172位用Asp-N蛋白酶(V162，promega)消化。用SYNAPT-G2-Si高分辨离子迁移率质谱仪(Waters)分析纯化GFP的分子质量。具体结果见图6。
本发明主要核酸元件的序列如下：
序列1 MmPylRS
序列2 MbPylRS
序列3 EcLeuRS
序列4 EcOMeYRS
序列5 tRNA1 MbPyl
序列6 tRNA2 MbPyl
序列7 tRNA MmPyl
序列8 tRNA EcLeu
序列9 tRNA1 EcTyr
序列10 tRNA2 EcTyr
序列11 GFP
序列12 RFP(DsRed2)
序列13 BFP(TagBFP)
以上介绍了本发明的较佳实施方式，旨在使得本发明的精神更加清楚和便于理解，并不是为了限制本发明，凡在本发明的精神和原则之内，所做的修改、替换、改进，均应包含在本发明所附的权利要求概括的保护范围之内。

Claims (7)

  1. 一种基因密码子拓展组合物，包括：
    (1)一种或多种氨基酰-tRNA合成酶，结合突变的tRNA；
    (2)一种或多种突变的tRNA，所述突变的tRNA反密码子环上突变为终止密码子的互补序列；
    其中，氨基酰-tRNA合成酶能将突变的tRNA结合到非天然氨基酸上产生氨基酰-tRNA；
    所述非天然氨基酸选自NAEK、Anap、pAcF；
    其包括以下三组氨基酰-tRNA合成酶与突变tRNA的组合：
    来自马氏古甲烷球菌的MmPylRs和tRNA MmPyl UCA的所述氨基酰-tRNA合成酶与突变tRNA组合；
    来自大肠杆菌的EcLeuRs和tRNA EcLeu CUA的所述氨基酰-tRNA合成酶与突变tRNA组合；
    以及
    来自大肠杆菌的EcTyrRs和tRNA EcTyr UUA的所述氨基酰-tRNA合成酶与突变tRNA组合。
  2. 一种在动物细胞中表达含有非天然氨基酸重组蛋白的方法，包括：
    (1)制备重组蛋白表达盒，其中重组蛋白编码序列中非天然氨基酸的密码子设置为终止密码子；
    (2)改造动物细胞，使其表达权利要求1中基因密码子拓展组合物中的氨基酰-tRNA合成酶和突变tRNA的组合；
    可选的，进一步突变eRF1以削弱eRF1与mRNA、和/或eRF1与核糖体的结合；
    (3)将步骤(1)中重组蛋白表达盒导入步骤(2)改造的动物细胞，制备重组动物细胞；
    (4)将步骤(3)构建的重组动物细胞在补充添加非天然氨基酸的培养基中培养，诱导含有非天然氨基酸重组蛋白的表达。
  3. 如权利要求2所述在动物细胞中表达含有非天然氨基酸重组蛋白的方法，其特征在于：
    (1)所述重组蛋白氨基酸序列中含有一种、两种或三种非天然氨基酸；或
    (2)在动物细胞中表达一种、两种或三种含有非天然氨基酸的重组蛋白，各重组蛋白中含有的非天然氨基酸种类相同或不同。
  4. 一种提高动物细胞中表达含非天然氨基酸重组蛋白通读率的方法，包括使突变的eRF1和含有PTC的mRNA在同一动物细胞内表达，其中，所述动物细胞中表达有权利要求1所述基因密码子拓展组合物中的氨基酰-tRNA合成酶和突变的tRNA；所述突变的eRF1削弱了eRF1与mRNA、和/或eRF1与核糖体的结合。
  5. 如权利要求4所述提高动物细胞中表达含非天然氨基酸重组蛋白通读率的方法，其中与野生型eRF1相比，所述突变的eRF1对mRNA、和/或核糖体具有较低的结合能力，并且所述突变的eRF1包括E55R、E55D、Y125F、N129P位点的突变。
  6. 如权利要求5所述提高动物细胞中表达含非天然氨基酸重组蛋白通读率的方法，其中突变的eRF1包括E55R突变体、E55D突变体、Y125F突变体、N129P突变体。
  7. 权利要求1所述基因密码子拓展组合物在重组表达含非天然氨基酸重组蛋白中的应用；其中所述重组表达以哺乳动物细胞为宿主，
    所述含非天然氨基酸重组蛋白包括：
    含有一个、两个或三个非天然氨基酸的一种重组蛋白，或者
    分别含有至少一个非天然氨基酸的两种或三种重组蛋白。
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