A kind of preparation method of protein containing non-natural amino acid

Abstract

The invention relates to a method for expressing recombinant proteins containing unnatural amino acids in animal cells. On the basis of four groups of aminoacyl-tRNA synthetases and corresponding tRNAs, aminoacyl-tRNAs that recognize stop codons are constructed, and three kinds of aminoacyl-tRNAs are obtained by screening. 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. To further improve the readthrough rate, the release factor eRF1 was also mutated to weaken its interaction with the stop 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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