Preparation method of protein containing non-natural amino acid

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...

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 acids in mammalian cells is low, especially when expressing polypeptides with multiple unnatural amino acid modification sites, different codon extension systems There will also be mutual interference between them, which not only affects the read-through efficiency, but also leads to the uncertainty of amino acid residues at the modification site

[0009] At present, in the prior art, 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

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