WO2023011486A1 - 高效引入赖氨酸衍生物的氨酰基-tRNA合成酶及其应用 - Google Patents

高效引入赖氨酸衍生物的氨酰基-tRNA合成酶及其应用 Download PDF

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WO2023011486A1
WO2023011486A1 PCT/CN2022/109759 CN2022109759W WO2023011486A1 WO 2023011486 A1 WO2023011486 A1 WO 2023011486A1 CN 2022109759 W CN2022109759 W CN 2022109759W WO 2023011486 A1 WO2023011486 A1 WO 2023011486A1
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trna synthetase
aminoacyl
fusion protein
host cell
lysine
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于歌
陈卫
吴松
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宁波鲲鹏生物科技有限公司
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • the invention relates to the field of biotechnology, in particular to an aminoacyl-tRNA synthetase for efficiently introducing lysine derivatives.
  • Aminoacyl-tRNA synthetases are enzymes involved in protein synthesis. Specifically, aminoacyl-tRNA can be synthesized by forming an ester bond between amino acid and tRNA. Aminoacyl-tRNA is a molecule that participates in the elongation of the peptide chains that make up proteins at the ribosome. Unnatural amino acids can be introduced into proteins using certain aminoacyl-tRNA synthetases. Currently, more than 30 complete proteins have been synthesized using aminoacyl-tRNA synthetase (aaRS)/tRNA pairs from various biological species.
  • aaRS aminoacyl-tRNA synthetase
  • TyrRS tyrosyl-tRNA synthetase
  • the pyrrolysyl-tRNA synthetase (CmaPylS) and the amber suppressor gene tRNAPyl from Candida methylophilus function as an orthogonal CmaPylS/CmaPylT pair in E. coli cells. function.
  • LysRS lysyl-tRNA synthetase
  • the present invention aims at improving the activity of the lysine derivatives by modifying the wild-type PylRS, and efficiently site-specifically integrating the lysine derivatives into the desired protein.
  • the object of the present invention is to provide an aminoacyl-tRNA synthetase for efficiently introducing lysine derivatives.
  • a mutant aminoacyl-tRNA synthetase is provided, the amino acid sequence of the mutant aminoacyl-tRNA synthetase is shown in SEQ ID NO.:3.
  • the mutant aminoacyl-tRNA synthetase is obtained by performing mutations on the basis of wild-type aminoacyl-tRNA synthetase.
  • amino acid sequence of the wild-type aminoacyl-tRNA synthetase is shown in SEQ ID NO.:1.
  • the wild-type aminoacyl-tRNA synthetase is derived from Candida methanophilus Candidatus Methanomethylophilus alvus.
  • the activity of the mutated aminoacyl-tRNA synthetase is at least 1.2 times, preferably at least 1.4 times, preferably at least 1.6, more preferably at least 2 times that of the wild-type aminoacyl-tRNA synthetase .
  • the aminoacyl-tRNA synthetase is a protein with the activity of combining unnatural amino acid to tRNA.
  • the unnatural amino acid is a lysine derivative or a lysine analogue.
  • the lysine derivatives include:
  • the unnatural amino acid is Boc-modified lysine.
  • the unnatural amino acid is butyneoxycarbonyl-modified lysine.
  • the ratio V1 of the expression level of the fusion protein to the expression level of the broken chain protein obtained by using the mutant aminoacyl-tRNA synthetase to express the fusion protein modified by the unnatural amino acid is the same as that obtained by using the wild-type aminoacyl-tRNA
  • V1/V2 is preferably not lower than 1.4, preferably not lower than 1.6, preferably not lower than Below 2.0, more preferably not below 2.4.
  • the chain-breaking protein is a non-target protein that does not contain unnatural amino acids produced during the expression of the fusion protein.
  • the fusion protein expression level L1 obtained by using a mutant aminoacyl-tRNA synthetase to express a non-natural amino acid modified fusion protein is the same as that obtained by using a wild-type aminoacyl-tRNA synthetase to perform non-natural amino acid modification.
  • L1/L2 is greater than 1.1, preferably greater than 1.2.
  • a vector containing the polynucleotide molecule described in the second aspect of the present invention there is provided a vector containing the polynucleotide molecule described in the second aspect of the present invention.
  • a host cell contains the vector according to the third aspect of the present invention or the polynucleotide molecule according to the second aspect of the present invention is chromosomally integrated.
  • the host cell is a prokaryotic cell, a eukaryotic cell or a mammalian cell.
  • the prokaryotic cell is Escherichia coli.
  • the host cell also contains a vector for expressing the fusion protein or a fusion protein expression cassette is integrated in the chromosome, wherein the fusion protein contains a lysine derivative.
  • the host cell further contains an expression vector of a fusion protein modified with tert-butoxycarbonyl (Boc).
  • the host cell further contains an expression vector for a butyneoxycarbonyl-modified fusion protein.
  • the target protein expressed by the host cell is human insulin and its derivatives, insulin aspart, insulin degludec precursor, insulin lispro, insulin glargine, insulin detemir precursor, parathyroid hormone , cortirelin, calcitonin, bivalirudin, glucagon-like peptide and its derivatives, exenatide, liraglutide precursor, semaglutide precursor, albiglutide Precursor, dulaglutide precursor, parathyroid hormone, ziconotide, sermorelin, somatorelin, secretin, teduglutide, hirudin, growth hormone, growth factor, growth hormone releasing factor , corticotropin, releasing factor, deserelin, desmopressin, elcatonin, glucagon, leuprolide, luteinizing hormone releasing hormone, somatostatin, thyrotropin releasing hormone Hormone, triptorelin, vasoactive intestinal peptide, interferon,
  • a method for preparing a fusion protein comprising the steps of:
  • the fusion protein includes a tert-butoxycarbonyl (Boc)-modified fusion protein and/or a butyneoxycarbonyl-modified fusion protein.
  • Boc tert-butoxycarbonyl
  • an enzyme preparation comprising the mutated aminoacyl-tRNA synthetase described in the first aspect of the present invention.
  • the seventh aspect of the present invention there is provided the use of the mutated aminoacyl-tRNA synthetase described in the first aspect of the present invention and the enzyme preparation described in the sixth aspect of the present invention, for preparing lysine-containing derivatives fusion protein.
  • FIG. 1 shows the map of pEvol-CmaPylS(wt)-pylT plasmid in Example 1.
  • FIG. 2 shows the map of pEvol-CmaPylS(mut)-pylT plasmid in Example 1.
  • Fig. 3 shows the map of the expression plasmid pBAD-FP-TEV-R-D of the insulin degludec precursor fusion protein in Example 2.
  • Figure 4 shows the electrophoretic pattern of the fusion protein expressed in Example 2.
  • lane 1 wild-type CmaPylS (wt);
  • lane 2 mutant CmaPylS (mut).
  • the present inventor obtained a mutant lysyl-tRNA synthetase unexpectedly through extensive and in-depth research and a large number of screenings.
  • the mutant lysyl-tRNA synthetase of the present invention has high activity and good solubility, and can significantly increase the amount of insertion of unnatural amino acids and the expression of target proteins containing unnatural amino acids, It reduces the expression level of chain-broken proteins that do not contain unnatural amino acids and facilitates the separation and purification of target proteins.
  • the inventors have completed the present invention.
  • wild-type lysyl-tRNA synthetase refers to a naturally occurring lysyl-tRNA synthetase that has not been artificially modified, and its nucleotides can be obtained by genetic engineering techniques, such as genome sequencing, Polymerase chain reaction (PCR), etc., the amino acid sequence can be deduced from the nucleotide sequence.
  • the source of the wild-type lysyl-tRNA synthetase is not particularly limited, preferably Candida methanophilus (Candidatus Methanomethylophilus alvus), but not limited thereto.
  • amino acid sequence of the wild-type lysyl-tRNA synthetase is shown in SEQ ID NO.:1.
  • the inventors of the present application have carried out a large number of screenings and obtained the mutant lysyl-tRNA synthetase whose amino acid sequence is shown in SEQ ID NO.: 3.
  • the mutation of the present invention Type lysyl-tRNA synthetase has high activity and good solubility, which can significantly increase the amount of unnatural amino acid insertion and the expression of fusion proteins containing unnatural amino acids, reduce the expression of broken chain proteins without unnatural amino acids and facilitate separation Purify and prepare the target protein.
  • the mutant lysyl-tRNA synthetase of the present invention also includes fragments, derivatives and analogs of mutants shown in SEQ ID NO.: 3, and described fragments, derivatives and analogs substantially maintain the mutant lysyl-tRNA synthetase of the present invention.
  • aminoacyl-tRNA synthetase which can be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide having a substituent in an amino acid residue, or (iii) a polypeptide of the invention fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused to a leader sequence, a secretory sequence, or a tag sequence such as 6His).
  • aminoacyl-tRNA synthetase which can be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide having a substituent in an amino acid residue, or (iii) a
  • a class of preferred active derivatives refers to that compared with the mutant shown in SEQ ID NO.: 3, there are at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is similar in nature or similar amino acids to form polypeptides.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also relates to a polynucleotide encoding the mutant lysyl-tRNA synthetase of the present invention.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • DNA can be either the coding strand or the non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence or a degenerate variant.
  • the full-length nucleotide sequence of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the mutant lysyl-tRNA synthetase (or its fragment, or its derivative) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells genetically engineered with the vectors of the present invention or the coding sequences of the present invention.
  • mutant lysyl-tRNA synthetase of the present invention can be chemically synthesized or recombinant.
  • the mutant lysyl-tRNA synthetase of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
  • a preferred method is to use recombinant technology to produce the mutant lysyl-tRNA synthetase of the present invention.
  • the polynucleotide of the present invention can be used to express or produce recombinant mutant lysyl-tRNA synthetase by conventional recombinant DNA technology. Generally speaking, there are the following steps:
  • the recombinant mutant lysyl-tRNA synthetase can be expressed in the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art.
  • Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the mutant lysyl-tRNA synthetase of the present invention has high activity and good solubility, and can significantly increase the amount of insertion of unnatural amino acids and the expression of target proteins containing unnatural amino acids.
  • mutant lysyl-tRNA synthetase of the present invention can reduce the expression of chain-broken proteins that do not contain unnatural amino acids and facilitate the separation and purification of target proteins.
  • the DNA sequence encoding CmaPylS was synthesized and cloned into the expression vector plasmid pEvol-pBpF (purchased from NTCC company, chloramphenicol resistance) SpeI-SalI site downstream of the araBAD promoter, wherein the SpeI restriction site is increased by PCR method, and the SalI site is owned by the vector itself.
  • the original glutamine promoter glnS of the expression vector plasmid pEvol-pBpF was retained.
  • the plasmid was extracted with a plasmid mini-extraction kit, and the plasmid was named pEvol-CmaPylS(wt)-pylT, and the plasmid map is shown in FIG. 1 .
  • mutant lysyl-tRNA synthetase CmaPylS (mut ), the amino acid sequence of which is shown below.
  • the plasmid pEvol-CmaPylS(mut)-pylT containing the coding sequence of the mutant lysyl-tRNA synthetase was constructed in the same way (the plasmid map is shown in Figure 2).
  • the plasmids pEvol-CmaPylS(wt)-pylT and pEvol-CmaPylS(mut)-pylT were respectively fused with the insulin degludec precursor to express the plasmid pBAD-FP-TEV-RD (constructed with reference to Chinese patent application 201910210102.9, and the plasmid map is shown in Figure 3 Shown) was co-transformed into E.coli Top10 competent cells by chemical method (CaCl method ) (competent cells were purchased from Thermo Company), and the transformed cells were cultured in a medium containing 25 ⁇ g/mL kanamycin and 17 ⁇ g /mL chloramphenicol on LB agar medium (10g/L yeast peptone, 5g/L yeast extract powder, 10g/L NaCl, 1.5% (g/mL) agar) and cultivate overnight at 37°C.
  • liquid LB medium (10 g/L yeast peptone, 5 g/L yeast extract powder, 10 g/L NaCl) containing 25 ⁇ g/mL kanamycin and 17 ⁇ g/mL chloramphenicol at 37 ° C, Grow overnight cultures at 220 rpm.
  • a feed medium containing 60% (ml/ml) glycerol and 250 g/L yeast peptone was added continuously until the end of fermentation. Cultivate until the OD 600 reaches 25-80, add L-arabinose at a final concentration of 0.25% (g/mL) for induction, continue to cultivate until the OD 600 reaches 180-220, put it into the tank, and then centrifuge (5000rpm, 30min, 25 °C) to collect the bacteria.
  • the expression of the fusion protein containing Boc-modified lysine in the whole cells of various strains was detected by SDS-polyacrylamide electrophoresis, and the electrophoretic pattern is shown in Figure 4.
  • the inclusion body obtained by expressing CmaPylS(mut) synthetase was dissolved in a 7.5M urea solution containing 2-10mM mercaptoethanol at pH 10.5, so that the concentration of the total protein after dissolution was 10-25 mg /mL. Dilute the sample by 5-10 times, and carry out conventional folding for 16-30 hours at 4-8° C. and pH 10.5-11.7. At 18-25°C, the pH value is maintained at 8.0-9.5, the fusion protein is hydrolyzed with trypsin and carboxypeptidase B for 10-20 hours, and then 0.45M ammonium sulfate is added to terminate the enzymolysis reaction.
  • the results of reverse phase HPLC analysis showed that the yield of this enzymolysis step was higher than 90%.
  • the insulin analog obtained after enzymolysis with trypsin and carboxypeptidase B is named Boc-insulin degludec precursor. Boc-insulin degludec precursor cannot be enzymatically degraded under the above conditions.
  • the sample was clarified by membrane filtration, and initially purified by hydrophobic chromatography with 0.45mM ammonium sulfate as a buffer, and the purity of SDS-polyacrylamide gel electrophoresis reached 90%.
  • Example 2 For each bacterial strain constructed in Example 2, carry out fermentation and culture under the same conditions, and when the OD600 reaches 25-80, add a final concentration of 0.25% L-arabinose and a final concentration of 5mM butynyloxycarbonyllysine Induction was carried out, and the culture was continued until the OD 600 reached 180-220, then placed in a tank, and then centrifuged (5000 rpm, 30 min, 25° C.) to collect the bacterial cells. The expression products were detected by SDS-polyacrylamide gel electrophoresis.

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Abstract

本发明提供了一种高效引入赖氨酸衍生物的突变型氨酰基-tRNA合成酶，其具有SEQ ID NO.:3所示氨基酸序列。相比野生型赖氨酰-tRNA合成酶，本发明突变型赖氨酰-tRNA合成酶活性高、可溶性好，可显著提高非天然氨基酸的插入量和含有非天然氨基酸的目的蛋白的表达量，可以降低不含非天然氨基酸的断链蛋白的表达量，易于分离纯化。本发明还提供了包含该突变型赖氨酰-tRNA合成酶的制剂和相关应用。

Description

高效引入赖氨酸衍生物的氨酰基-tRNA合成酶及其应用 技术领域
本发明涉及生物技术领域，具体地涉及高效引入赖氨酸衍生物的氨酰基-tRNA合成酶。
背景技术
氨酰基-tRNA合成酶(aaRS)是参与蛋白质合成的酶。具体而言，可使氨基酸与tRNA形成酯键从而具有合成氨酰基-tRNA的活性。氨酰基-tRNA是一种分子，参与在核糖体构成蛋白质的肽链的伸长。使用某些氨酰基-tRNA合成酶可将非天然氨基酸引入蛋白质中，目前使用源自各种生物种类的氨基酰-tRNA合成酶(aaRS)/tRNA对，已经合成了30种以上的全蛋白质。历史最长并且应用于许多用非天然氨基酸导入的体系为酪氨酰-tRNA合成酶(TyrRS)突变体与经琥珀抑制基因化tRNA Tyr对。就该方法而言，成为关键的是其正交的关系，即，真细菌、与古细菌和真核生物这两个组中的aaRS在各自的组内将tRNA氨基酰化，但不能将其它组的tRNA氨基酰化。
另一方面，来自噬甲基念珠菌(Candidatus Methanomethylophilus alvus Mx1201)的吡咯赖氨酰-tRNA合成酶(CmaPylS)和琥珀抑制基因tRNAPyl，在大肠杆菌细胞内作为具有正交性的CmaPylS/CmaPylT对而发挥功能。
通常，为了将赖氨酸衍生物导入蛋白质，存在改变赖氨酰-tRNA合成酶(LysRS)的底物特异性的方法。但是，由于LysRS对赖氨酸的识别严密，因此，至今为止，很难将具有各种大小、形状的官能团的赖氨酸衍生物位点特异性地导入蛋白质。吡咯赖氨酸(Pyrrolysine)是在侧链上具有大体积甲基吡咯啉部分的赖氨酸衍生物。野生型Pyrrolysyl-tRNA合成酶(PylRS)虽能够活化其赖氨酸衍生物，但是其能够活化的赖氨酸衍生物的活力有限。
因此，亟需发明一种新的能够高效引入赖氨酸衍生物的氨酰基-tRNA合成酶。
发明内容
本发明致力于通过改造野生型PylRS，增强其对于赖氨酸衍生物的活性，高效地将赖氨酸衍生物位点特异性的整合在希望的蛋白质中。
本发明的目的在于提供一种高效引入赖氨酸衍生物的氨酰基-tRNA合成酶。
在本发明的第一方面，提供了一种突变的氨酰基-tRNA合成酶，所述突变的氨酰基-tRNA合成酶的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述突变的氨酰基-tRNA合成酶是在野生型氨酰基-tRNA合成酶的基础上进行突变获得的。
在另一优选例中，所述野生型氨酰基-tRNA合成酶的氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述的野生型氨酰基-tRNA合成酶来源于嗜甲烷假丝酵母Candidatus Methanomethylophilus alvus。
在另一优选例中，所述突变的氨酰基-tRNA合成酶的活性是野生型氨酰基-tRNA合成酶的至少1.2倍，优选地至少1.4倍，优选地至少1.6，更优选地至少2倍。
在另一优选例中，所述的氨酰基-tRNA合成酶是具有将非天然氨基酸结合于tRNA的活性的蛋白质。
在另一优选例中，所述的非天然氨基酸为赖氨酸衍生物，赖氨酸类似物。
在另一优选例中，所述的赖氨酸衍生物包括：
·包含芳香族侧链；
·包含叠氮基；
·包含炔烃基团；或者
·包含醛或酮基团的脂肪酰化赖氨酸。
在另一优选例中，所述的非天然氨基酸为Boc修饰的赖氨酸。
在另一优选例中，所述的非天然氨基酸为丁炔氧羰基修饰的赖氨酸。
在另一优选例中，利用突变的氨酰基-tRNA合成酶进行非天然氨基酸修饰的融合蛋白的表达所得到的融合蛋白表达量与断链蛋白表达量比值V1，与利用野生型氨酰基-tRNA合成酶进行非天然氨基酸修饰的融合蛋白的表达所得到的融合蛋白表达量与断链蛋白表达量比值V2相比，V1/V2优选地不低于1.4，优选地不低于1.6，优选地不低于2.0，更优选地不低于2.4。
在另一优选例中，所述的断链蛋白是在融合蛋白表达过程中产生的不含非天然氨基酸的非目的蛋白。
在另一优选例中，利用突变的氨酰基-tRNA合成酶进行非天然氨基酸修饰的融合蛋白的表达所得到的融合蛋白表达量L1，与利用野生型氨酰基-tRNA合成酶进行非天然氨基酸修饰的融合蛋白的表达所得到的融合蛋白表达量L2相比，L1/L2大于1.1，优选地大于1.2。
在本发明的第二方面，提供了一种多核苷酸分子，所述多核苷酸分子编码本发明第一方面所述的突变的氨酰基-tRNA合成酶。
在本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸分子。
在本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体或染色体整合有本发明第二方面所述的多核苷酸分子。
在另一优选例中，所述宿主细胞为原核细胞、真核细胞或哺乳细胞。
在另一优选例中，所述原核细胞为大肠杆菌。
在另一优选例中，所述宿主细胞还含有用于表达融合蛋白的载体或染色体中整合有融合蛋白表达盒，其中，所述的融合蛋白中含有赖氨酸衍生物。
在另一优选例中，所述宿主细胞还含有叔丁氧羰基(Boc)修饰的融合蛋白的表达载体。
在另一优选例中，所述宿主细胞还含有丁炔氧羰基修饰的融合蛋白的表达载体。
在另一优选例中，所述宿主细胞表达的目的蛋白为人胰岛素及其衍生物、门冬胰岛素、德谷胰岛素前体、赖脯胰岛素、甘精胰岛素、地特胰岛素前体、甲状旁腺素、可的瑞琳、降血钙素、比伐卢定、胰高血糖素样肽及其衍生物、艾塞那肽、利拉鲁肽前体、索马鲁肽前体、阿必鲁肽前体、度拉鲁肽前体、甲状旁腺激素、齐考诺肽、舍莫瑞林、生长瑞林、分泌素、替度鲁肽、水蛭素、生长激素、生长因子、生长激素释放因子、促肾上腺皮质激素、释放因子、德舍瑞林、去氨加压素、依降钙素、胰高血糖素、亮丙瑞林、促黄体激素释放激素、生长激素抑制素、促甲状腺激素释放激素、曲普瑞林、血管活性肠肽、干扰素、BH3肽、淀粉样变肽，或上述肽的片段，或其组合。
在本发明的第五方面，提供了一种制备的融合蛋白的方法，包括步骤：
(i)在适合的条件下，培养本发明第四方面所述的宿主细胞，从而表达出所述的融合蛋白；和
(ii)分离所述的融合蛋白，
其中，所述的融合蛋白中含有赖氨酸衍生物。
在另一优选例中，所述的融合蛋白包括叔丁氧羰基(Boc)修饰的融合蛋白和/或丁炔氧羰基修饰的融合蛋白。
在本发明的第六方面，提供了一种酶制剂，所述酶制剂包含本发明第一方面所述的突变的氨酰基-tRNA合成酶。
在本发明的第七方面，提供了如本发明第一方面所述的突变的氨酰基-tRNA合成酶、本发明第六方面所述的酶制剂的用途，用于制备含有赖氨酸衍生物的融合蛋白。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了实施例1中pEvol-CmaPylS(wt)-pylT质粒的图谱。
图2显示了实施例1中pEvol-CmaPylS(mut)-pylT质粒的图谱。
图3显示了实施例2中德谷胰岛素前体融合蛋白表达质粒pBAD-FP-TEV-R-D的图谱。
图4显示了实施例2中表达的融合蛋白的电泳图谱。其中，泳道1：野生型CmaPylS(wt)；泳道2：突变型CmaPylS(mut)。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，意外地获得一种突变型赖氨酰-tRNA合成酶。相比野生型赖氨酰-tRNA合成酶，本发明突变型赖氨酰-tRNA合成酶活性高、可溶性好，可显著提高非天然氨基酸的插入量和含有非天然氨基酸的目的蛋白的表达量、降低不含非天然氨基酸的断链蛋白的表达量且有利于分离纯化目的蛋白。在此基础上，发明人完成了本发明。
野生型赖氨酰-tRNA合成酶
如本文所用，“野生型赖氨酰-tRNA合成酶”是指天然存在的、未经过人工改造的赖氨酰-tRNA合成酶，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚 合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型赖氨酰-tRNA合成酶的来源没有特别限制，优选的为来源嗜甲烷假丝酵母(Candidatus Methanomethylophilus alvus)，但不限于此。
在本发明的一个优选例中，所述野生型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:1所示。
Figure PCTCN2022109759-appb-000001
突变的赖氨酰-tRNA合成酶
本申请的发明人进行了大量的筛选，获得了氨基酸序列如SEQ ID NO.:3所示的突变型赖氨酰-tRNA合成酶，相比野生型赖氨酰-tRNA合成酶，本发明突变型赖氨酰-tRNA合成酶活性高、可溶性好，可显著提高非天然氨基酸插入量和含有非天然氨基酸的融合蛋白的表达量、降低不含非天然氨基酸的断链蛋白的表达量且利于分离纯化制备目的蛋白。
本发明的突变型赖氨酰-tRNA合成酶还包括SEQ ID NO.:3所示突变体的片段、衍生物和类似物，所述的片段、衍生物和类似物基本保持本发明突变型赖氨酰-tRNA合成酶的活性，其可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)本发明多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与SEQ ID NO.:3所示突变体相比，有至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
编码序列
本发明还涉及编码本发明突变型赖氨酰-tRNA合成酶的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与编码区序列相同或者是简并的变异体。本发明的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明突变型赖氨酰-tRNA合成酶(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明的编 码序列经基因工程产生的宿主细胞。
制备方法
本发明的突变型赖氨酰-tRNA合成酶可以是化学合成的，或重组的。相应地，本发明的突变型赖氨酰-tRNA合成酶可用常规方法人工合成，也可用重组方法生产。
一种优选的方法是用重组技术产生本发明的突变型赖氨酰-tRNA合成酶。通过常规的重组DNA技术，可利用本发明的多核苷酸来表达或生产重组的突变型赖氨酰-tRNA合成酶。一般来说有以下步骤：
(1)用本发明突变型赖氨酰-tRNA合成酶的多核苷酸(或变异体)编码，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
重组的突变型赖氨酰-tRNA合成酶可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点在于：
(a)本发明的突变型赖氨酰-tRNA合成酶活性高、可溶性好，可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白的表达量。
(b)本发明的突变型赖氨酰-tRNA合成酶可以降低不含非天然氨基酸的断链蛋白的表达量且利于分离纯化目的蛋白。
实施例1 野生型赖氨酰-tRNA合成酶及其突变体的构建
根据赖氨酰-tRNA合成酶CmaPylS的氨基酸序列1(SEQ ID NO.:1)，及大肠杆菌的密码子偏好性，合成了编码CmaPylS的DNA序列，克隆至表达载体质粒pEvol-pBpF(购自NTCC公司，氯霉素抗性)的araBAD启动子下游SpeI-SalI位点，其中SpeI酶切位点通过PCR方法增加，SalI位点为载体本身具有。保留表达载体质粒pEvol-pBpF原有的谷氨酰胺启动子glnS。在表达载体质粒pEvol-pBpF的proK启动子下 游，以PCR方法插入赖氨酰-tRNA合成酶的tRNA(CmaPylT)的DNA序列，具体序列如下：
Figure PCTCN2022109759-appb-000002
以化学法(CaCl 2法)转化至E.coli Top10感受态细胞中，将所述转化的细胞培养在含有17μg/mL氯霉素的LB琼脂培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl，1.5％(g/mL)琼脂)上，37℃培养过夜。挑取单个活菌落，在含有氯霉素的液体LB培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl)中37℃、220rpm培养过夜。以质粒小量提取试剂盒提取质粒，该质粒命名为pEvol-CmaPylS(wt)-pylT，质粒图谱如图1所示。
以野生型赖氨酰-tRNA合成酶的氨基酸序列为基础，对其进行突变改造，获得表达情况显著改善的如SEQ ID NO.:3所示的突变型赖氨酰-tRNA合成酶CmaPylS(mut)，其氨基酸序列如下所示。
Figure PCTCN2022109759-appb-000003
以相同的方法构建包含突变型赖氨酰-tRNA合成酶编码序列的质粒pEvol-CmaPylS(mut)-pylT(质粒图谱如图2所示)。
实施例2 双质粒表达菌株构建和叔丁氧羰基(Boc)修饰融合蛋白的高密度表达
将质粒pEvol-CmaPylS(wt)-pylT和pEvol-CmaPylS(mut)-pylT分别与德谷胰岛素前体融合蛋白表达质粒pBAD-FP-TEV-R-D(参照中国专利申请201910210102.9构建，质粒图谱如图3所示)以化学法(CaCl 2法)共转化到E.coli Top10感受态细胞中(感受态细胞购买自Thermo公司)，将所述转化的细胞培养在含有25μg/mL卡那霉素和17μg/mL氯霉素的LB琼脂培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl，1.5％(g/mL)琼脂)上37℃培养过夜。挑取单个活菌落，在含有25μg/mL卡那霉素和17μg/mL氯霉素的液体LB培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl)中37℃、220rpm培养过夜培养。
将各菌种接种于液体LB培养基中37℃、220rpm培养过夜，以1％(ml/ml)接种罐发酵培养基(12g/L酵母蛋白胨，24g/L酵母浸粉，4mL/L甘油，12.8g/L磷酸氢二钠，3g/L磷酸二氢钾，0.3‰(ml/ml)消泡剂(江苏赛欧信越消泡剂有限公司)，5mM Boc-Lys)，在35(±3)℃，200～1000rpm，空气流量2～6L/min的条件下培养。培养3～10h后，流加含60％(ml/ml)甘油和250g/L酵母蛋白胨的补料培养基，持续至发酵结束。培养至OD 600达到25～80时，加入终浓度为0.25％(g/mL)L-阿拉伯糖进行诱导，继续培养至OD 600达到180～220时，放罐，然后离心(5000rpm，30min，25℃)收集菌体。以SDS-聚丙烯酰胺电泳对各菌种全细胞中含有Boc修饰赖氨酸的融合蛋白表达情况进行检测，其电泳图谱如图4所示。
结果显示，合成酶CmaPylS(wt)和合成酶CmaPylS(mut)蛋白大部分表达在周质上清，极少量表达为包涵体。表达产物中的融合蛋白(目标蛋白)和断链蛋白是以不可溶的“包涵体”形式表达。为释放包涵体，用高压均质机将大肠杆菌细胞破碎。合成酶和目标蛋白分别处在上清及包涵体中,易于分离纯化。用离心方式进行固液分离，去除核酸、细胞碎片和可溶性蛋白。用纯化水洗涤含有融合蛋白的包涵体，将所得的包涵体沉淀用作折叠的原材料。
不同突变酶的融合蛋白表达量如表1所示。
表1
Figure PCTCN2022109759-appb-000004
为了使融合蛋白重折叠，将利用CmaPylS(mut)合成酶表达获得的包涵体溶解于pH10.5并含有2～10mM巯基乙醇的7.5M脲溶液中，使溶解后总蛋白的浓度为10～25mg/mL。将样品稀释5～10倍，在4～8℃，pH为10.5～11.7的条件下进行常规折叠16～30小时。于18～25℃下，pH值维持在8.0～9.5，用胰蛋白酶和羧肽酶B将融合蛋白酶解10～20小时，然后加入0.45M硫酸铵终止酶解反应。
反相HPLC分析结果表明，该酶解步骤的收率高于90％。胰蛋白酶与羧肽酶B酶解后获得的胰岛素类似物被命名为Boc-德谷胰岛素前体。Boc-德谷胰岛素前体在上述条件下不能被酶解。通过膜过滤澄清样品，以0.45mM硫酸铵作为缓冲液，经疏水层析初纯化，SDS-聚丙烯酰胺凝胶电泳纯度达90％。并且对获得的Boc-德谷胰岛素前体进行MALDI-TOF质谱分析，结果检测出其分子量与理论分子量5907.7Da相符合。经疏 水层析洗脱收集样品，经化学修饰和两步高压反相层析，得到德谷胰岛素。
实施例3 丁炔氧羰基修饰融合蛋白的高密度表达
对实施例2中构建的各菌株，以相同的条件进行发酵培养，培养至OD 600达到25～80时，加入终浓度为0.25％L-阿拉伯糖和终浓度为5mM丁炔氧羰基赖氨酸进行诱导，继续培养至OD 600达到180～220时，放罐，然后离心(5000rpm，30min，25℃)收集菌体。以SDS-聚丙烯酰胺凝胶电泳检测表达产物。
结果显示，合成酶CmaPylS(wt)和合成酶CmaPylS(mut)蛋白大部分表达在周质上清，极少量表达为包涵体。表达产物中的融合蛋白(目标蛋白)和断链蛋白是以不可溶的“包涵体”形式表达。为释放包涵体，用高压均质机将大肠杆菌细胞破碎。合成酶和目标蛋白分别处在上清及包涵体中，易于分离纯化。用离心方式进行固液分离，去除核酸、细胞碎片和可溶性蛋白。用纯化水洗涤含有融合蛋白的包涵体，所得的包涵体沉淀用作折叠的原材料。
不同突变酶的融合蛋白表达量如表2所示。
表2
Figure PCTCN2022109759-appb-000005
结果表明，利用本发明的突变酶制备含有丁炔氧羰基赖氨酸的目的蛋白，可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白的表达量、降低不含非天然氨基酸的断链蛋白的表达量且利于分离纯化目的蛋白。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种突变的氨酰基-tRNA合成酶，其特征在于，所述突变的氨酰基-tRNA合成酶的氨基酸序列如SEQ ID NO.:3所示。
  2. 如权利要求1所述的突变的氨酰基-tRNA合成酶，其特征在于，所述的氨酰基-tRNA合成酶是具有将非天然氨基酸结合于tRNA的活性的蛋白质。
  3. 如权利要求2所述的突变的氨酰基-tRNA合成酶，其特征在于，所述非天然氨基酸为赖氨酸衍生物或赖氨酸类似物。
  4. 如权利要求3所述的突变的氨酰基-tRNA合成酶，其特征在于，所述的赖氨酸衍生物包括：
    ·包含芳香族侧链；
    ·包含叠氮基；
    ·包含炔烃基团；或者
    ·包含醛或酮基团的脂肪酰化赖氨酸。
  5. 如权利要求3所述的突变的氨酰基-tRNA合成酶，其特征在于，所述的非天然氨基酸为Boc修饰的赖氨酸。
  6. 如权利要求3所述的突变的氨酰基-tRNA合成酶，其特征在于，所述的非天然氨基酸为丁炔氧羰基修饰的赖氨酸。
  7. 一种多核苷酸分子，其特征在于，所述多核苷酸分子编码权利要求1所述的突变的氨酰基-tRNA合成酶。
  8. 一种载体，其特征在于，所述载体含有权利要求7所述的多核苷酸分子。
  9. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求8所述的载体或染色体整合有权利要求7所述的多核苷酸分子。
  10. 如权利要求9所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌。
  11. 如权利要求9所述的宿主细胞，其特征在于，所述宿主细胞还含有用于表达融合蛋白的载体或染色体中整合有融合蛋白表达盒，其中，所述的融合蛋白中含有赖氨酸衍生物。
  12. 如权利要求11所述的宿主细胞，其特征在于，所述宿主细胞表达的目的蛋白为人胰岛素及其衍生物、门冬胰岛素、德谷胰岛素前体、赖脯胰岛素、甘精胰岛素、地特胰岛素前体、甲状旁腺素、可的瑞琳、降血钙素、比伐卢定、胰高血糖素样肽及其衍生物、艾塞那肽、利拉鲁肽前体、索马鲁肽前体、阿必鲁肽前体、度拉鲁肽前体、 甲状旁腺激素、齐考诺肽、舍莫瑞林、生长瑞林、分泌素、替度鲁肽、水蛭素、生长激素、生长因子、生长激素释放因子、促肾上腺皮质激素、释放因子、德舍瑞林、去氨加压素、依降钙素、胰高血糖素、亮丙瑞林、促黄体激素释放激素、生长激素抑制素、促甲状腺激素释放激素、曲普瑞林、血管活性肠肽、干扰素、BH3肽、淀粉样变肽，或上述肽的片段，或其组合。
  13. 一种制备融合蛋白的方法，其特征在于，包括步骤：
    (i)在适合的条件下，培养权利要求9所述的宿主细胞，从而表达出所述融合蛋白；和
    (ii)分离所述融合蛋白，
    其中，所述的融合蛋白中含有赖氨酸衍生物。
  14. 如权利要求13所述的方法，其特征在于，所述的融合蛋白包括叔丁氧羰基(Boc)修饰的融合蛋白和/或丁炔氧羰基修饰的融合蛋白。
  15. 一种酶制剂，其特征在于，所述酶制剂包含权利要求1所述的突变的氨酰基-tRNA合成酶。
  16. 如权利要求1所述的突变的氨酰基-tRNA合成酶、权利要求15所述的酶制剂的用途，其特征在于，用于制备含有赖氨酸衍生物的融合蛋白。
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