Preparation method of protein containing non-natural amino acid

An unnatural amino acid and aminoacyl technology, applied in the field of biopharmaceuticals, can solve problems such as the lack of research methods for small molecule drug systems, the reduction of drug efficacy caused by neutralizing antibodies, and the modification of multiple proteins with different unnatural amino acids

Active Publication Date: 2021-11-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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; ②Large molecular weight and strong immunogenicity, the body injection induces the production of neutralizing antibodies in the body and the drug effect continues to decrease; ③It cannot be modified at a fixed point, and lacks systematic research methods for structure-activity relationships like small molecule drugs, etc.
[0008] The large-scale expression and modification of protein drugs are still mainly carried out in bacteria, and the insertion efficiency of unnatural amino a

Method used

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  • Preparation method of protein containing non-natural amino acid
  • Preparation method of protein containing non-natural amino acid
  • Preparation method of protein containing non-natural amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Four kinds of unnatural amino acid systems expanded into 12 different systems

[0087] We used available complexes of Desulfobacillus hafniense except for pylMmRS (PDB:2ZIM), two synthetases that are structurally conserved. According to the binding structure of synthetase and corresponding tRNA, tRNA Pyl and tRNA Leu The anticodon loop of is not bound by its synthetase. The anticodon loop of tRNATyr does form hydrogen bonds with D286 and C231 in TyrRS, but the mutated anticodons UUA, CUA, and UCA maintain weak interactions with the aforementioned key residues in TyrRS, thus ensuring successful aminoacylation (see figure 1 ).

[0088] In order to construct plasmids encoding four aminoacyl tRNA synthetases (OMeYRS, EcleuRS, MbPylRS and MmpylRS), the corresponding genes expressed under the CMV promoter were inserted into the pcDNA3.1(+) vector by double digestion reaction. tRNA MbPyl and tRNA EcTyr Expressed by H1 and U6 promoters, tRNA Ecleu Expressed by ...

Embodiment 2

[0095] Example 2 Orthogonality verification of four unnatural amino acid systems and screening of the best combination of the three systems with the highest efficiency in mammalian cells

[0096] To rule out interactions of different systems, we verified the mutual orthogonality of the four available aaRS / tRNA systems ( Figure 3C ).

[0097] First, we performed orthogonality verification at the UAA and aaRS / tRNA pair levels. 48 hours after the transfection system and the corresponding GFP plasmid, different unnatural amino acids were added to the culture medium, the fluorescence of GFP was observed, and the fluorescence intensity was compared by flow cytometric analysis. The results are as follows Figure 3A shown.

[0098] We then determined orthogonality at the level of synthetases and tRNAs. Synthetases were co-transfected with different tRNAs CUA In the HEK293T cells, as well as the GFP containing the TAG mutation, observe the fluorescence of GFP, and perform flow cyt...

Embodiment 3

[0106] Example 3 Plasmid construction of RFP, BFP, GFP and controllable expression of three fluorescent proteins in mammalian cells

[0107] We first constructed the RFP based on point mutation and molecular cloning methods 36TGA 、BFP 39TAG and GFP 39TAA Mutants, primers for constructing the above three mutant fluorescent proteins are shown in Table 5.

[0108] Table 5. Primer sequences used in the construction of three fluorescent protein mutants

[0109] Primer sequence GFP 39TAA -F

[0110] System plasmid containing Mmpyl-tRNA pyl UCA ,EcTyr-tRNA Tyr UUA , Ecleu-tRNA leu CUA , transfected them together into HEK293T cells, designed 8 groups of experiments, added 0, 1, 2, and 3 different unnatural amino acids to the medium, collected the cells after 48 hours, and extracted the protein for Western blotting. The expression of three fluorescent proteins was detected under different experimental conditions. For specific results, see Figure 4 .

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Abstract

The invention relates to a method for expressing recombinant protein containing unnatural amino acid in animal cells, which comprises the following steps: on the basis of four groups of aminoacyl-tRNA synthetases and corresponding tRNA, constructing aminoacyl-tRNA for identifying termination codons, and screening to obtain three gene codon expansion systems. The three gene codon expansion systems are high in termination codon reading efficiency and high in mutual compatibility, so that three non-natural amino acids can be inserted into one foreign protein, or different non-natural amino acids can be inserted into three foreign proteins of the same cell respectively. In order to further improve the readthrough rate, the release factor eRF1 is mutated to weaken the interaction between the release factor eRF1 and the termination codon.

Description

technical field [0001] 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. Background technique [0002] There are a total of 64 triplet codons in nature. In general organisms, 61 codons encode 20 natural amino acids, while the other three codons (UAA, UGA, UAG) do not encode any amino acids. When the ribosome When translation reaches these codons, there will be normal termination factors to terminate protein translation. As more biological phenomena were revealed, scientists found that in archaea, one of the stop codons, the amber stop codon UAG, was used to encode special amino acids into essential proteins to ensure the normal growth of bacteria. Based on this, the scientists took out the tRNA and tRNA synthetase pa...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/11C12N15/85C12N15/65C07K14/47
CPCC12N9/93C12N15/11C12N15/85C12N15/65C07K14/4702C12Y601/01004C12Y601/01001C12Y601/01026C12N2800/22C12N2830/36C12N2800/107
Inventor 夏青史宁宁
Owner PEKING UNIV
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