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Advancing the Genetic Code: Incorporating Novel Amino Acids to Enhance Protein Engineering Capabilities

Researchers in synthetic biology have innovatively repurposed infrequently used codons to integrate novel amino acids into proteins, enhancing the field of protein engineering.

Living organisms naturally produce a vast array of proteins by arranging 20 standard amino acids in various sequences and lengths. Historically, attempts to broaden this range by introducing synthetic amino acids into proteins have seen limited success, mainly because cells incorporated these additional components into only a small fraction of the target proteins.

A recent study published in Science presents a groundbreaking approach for embedding new amino acids into proteins. This technique offers a new pathway to generate proteins with unique properties in higher quantities, significantly advancing synthetic biology.

James Van Deventer, a protein engineer at Tufts University who was not involved in the research, commented, “The team achieved over 80 percent efficiency, demonstrating their thorough commitment to this work.”

In protein synthesis, ribosomes follow genetic instructions conveyed through RNA transcripts, which outline the sequence of the 20 amino acids. RNA is composed of just four bases—adenine (A), cytosine (C), guanine (G), and uracil (U). To code for the 20 amino acids, ribosomes interpret these bases in groups of three, known as codons, of which there are 64 different types. Transfer RNA (tRNA) molecules match these codons to specific amino acids and assist in building the protein chain. Out of the 64 codons, three are stop codons that signal the end of the protein synthesis process by instructing the ribosome to release the completed polypeptide chain.

In earlier efforts to broaden the genetic code, researchers attempted to introduce a transfer RNA (tRNA) that was attached to a non-standard amino acid and could recognize a stop codon. However, this approach was largely unsuccessful because the proteins responsible for signaling the end of protein synthesis had a stronger affinity for the stop codon than the modified tRNA. As a result, the stop codons typically maintained their original function. “The efficiency of this method was generally below five percent, making it impractical for most applications,” explained Shixian Lin, a synthetic biologist from Zhejiang University and coauthor of the study. Lin proposed that using a tRNA designed to pair with a rare codon might be more effective. In cases where codons are infrequent, the cell produces fewer of the corresponding tRNA, thereby reducing competition and potentially increasing efficiency.

Optimizing Rare Codon Usage: Enhancing Protein Engineering with Targeted tRNA Approaches

First, Lin’s team needed to identify rare codons in human cell lines. They used RNA sequencing to identify the seven least frequently used codon triplets. To find out which of these rare codons could be most effectively utilized, they incorporated each of the seven codons into the gene for enhanced green fluorescent protein (eGFP) and introduced this modified gene into the cells. They then treated the cell cultures with synthetic tRNA, which carried a distinct, traceable non-standard amino acid and was designed to recognize these rare codons. The researchers discovered that the TCG codon resulted in the highest incorporation of the new amino acid into the protein.

Since the TCG codon effectively directed the ribosome to add the non-standard amino acid to the desired protein, the scientists hypothesized that it might also incorporate this new amino acid into other cellular proteins containing the rare codon, potentially affecting their functions. To evaluate unintended background incorporation, they exposed cells to different tRNAs carrying a traceable amino acid, each recognizing either the TCG codon or one of the other six rare codons. After extracting and staining all the cellular proteins for the traceable amino acid, they found that the TCG-specific tRNA yielded the weakest signal, indicating the lowest level of non-specific incorporation into other proteins.

To determine why the TCG codon had minimal impact on other proteins, Lin’s team analyzed the RNA sequences flanking the TCG codon. They found that the efficiency of incorporating the non-standard amino acid was influenced by the sequences surrounding the codon. “We discovered that even a single nucleotide polymorphism upstream of the codon can significantly alter the recoding efficiency,” Lin explained. This finding suggests that researchers should carefully position the rare codon within the sequence. “In some cases, moving these rare codons to different locations within the protein can cause the recoding efficiency to vary dramatically, ranging from five percent to 99 percent,” he added.

 

Source: https://www.sciencedirect.com/science/article/abs/pii/S0022283621006197

 

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