In a very recent paper published on Nature Biotechnology, a group of Researchers at The University of Texas at Austin described a method to make therapeutic proteins more stable. They did this by replacing cysteine with another amino acid called selenocysteine in the protein. This non-standard amino acid (ncAA) form hardier chemical bonds, which could lead to drugs with increased stability.
The team demonstrated the practical application of this method by producing the functional region of the breast cancer drug Herceptin®. The ncAA containing proteins survive longer in the human body compared with wild type proteins, demonstrating great potential for more stable drugs with increased effectiveness.
A pharmaceutical company, GRO Biosciences, is already using the new method. "In all instances, according to GRO Biosciences, yields were high, selenocysteine incorporation at the desired sites was 100%, and all diselenide bonds formed correctly, leading to the properly folded protein. The diselenide bonds dramatically increased stability in physiologically relevant conditions, as confirmed by functional assays."
George M. Church, professor of genetics at Harvard Medical School is also one of a cofounders: "GRO Biosciences' technology addresses the fundamental limitations of producing proteins with nonstandard amino acids, opening up the possibility of creating a new universe of designer proteins with enhanced therapeutic properties at commercial scale."
How could the team engineer a cell to incorporate ncAA? This could be a long story.
When making proteins, cells read the code of DNA in groups of three called "codons" in the process of translation. The genetic code consists of 64 triplets of nucleotides called codons. With the exception of three stop codons, each codon encodes for one of the 20 canonical amino acids and most of the amino acids are encoded by more than one codon. For example, ACU, ACC, ACA and ACG all code for threonine.
What do you get from this? This language of life is redundant, so two thirds of the triplet codes can theoretically be reassigned to a novel amino acid. This redundancy gives scientists the possibility of redefine the function of a codon to a nonstandard amino acid.
How can we redefine the function of a “fixed” codon? One initial thing is that we will need a tRNA:ncAA pair that tells the cell which amino acid to choose to incorporate into the protein.
Early work has focused on the stop signals, TAA, TAG, and TGA, which don’t already encode any amino acids and are thus available for recoding. A stop signal can be converted to a “sense” signal if a transfer RNA (tRNA) molecule is available to decode it. This was demonstrated by the same team in 2015. The researchers engineered a tRNA(Sec) that is compatible with the canonical translation machinery to incorporate selenocysteine by sensing TGA.
However, the release factor normally signaled by the codon to terminate translation still recognizes TGA and competes with the engineered tRNA, reducing the amount of protein that can be generated with the desired amino acid.
Is it possible to delete RF1 and make the cell “naturally” think a stop codon actually encode an ncAA? For this goal, a group of scientist in George M. Church lab took a very ambitious move. They wanted to completely free up one of these redundant three-letter codons and in an organism. They recoded the E. coli genome by replacing every instance of TAG in the genetic code with TAA. In the recoded E. coli, the TAG codon was no longer necessary, so the cellular machinery responsible for the TAG stop function can be deleted. This resulted in an unused TAG codon that was free to be reassigned to a new function.
Completed replacing all TAG in the E. coli requires 314 modifications in the genome. How did the team achieve this?
The recoding project started by developing two powerful genome engineering technology ten years ago.
The first technology is called MAGE, which stands for "multiplex automated genome engineering". It allowed scientist to introduce tens of changes to a bacterial genome nearly simultaneously. The DNA corresponding to the desired changes is mixed and introduced into the bacteria on a custom-made machine.
TAG appears in 314 places throughout the E.coli genome as a stop codon. For each one, the team created a small stretch of DNA that had TAA instead of TAG. They fed these editing fragments into bacteria, which used them to build new copies of their own DNA. In this way, the team created 32 strains of E. coli that each containing a segment with all TAG substituted by TAA.
The next step is to combine the 32 strains into a single strain? For this, the team developed CAGE (conjugative assembly genome engineering) technology, which draws on bacteria’s natural ability to swap genetic material. This method resembles a playoff bracket — a hierarchy that combine 16 pairs to eight to four to two to one. One strain of each pair would deliver its edited genes into its partner, and the incoming genes were designed to merge with those of the recipient in specific ways.
With TAG free from its duties as a punctuation mark, the team could delete release factor 1 (RF1) and reassign TAG to new amino acids, just as they planned. “In a plug and play manner, you can start to pop in new amino acids with new chemistries,” says Isaacs, the key team member, now assistant professor in Yale.
Besides incorporating ncAA, the recoded organism have other important applications. One is to make cells resistant to viruses. Viruses make copies of themselves by hijacking the protein-making factories of their hosts. So if their hosts use a different set of genetic code, their factories will not be able to make the viruses proteins. The researches already observed enhanced resistance to bacteriophage T7 in the recoded E. coli.
Another application is to prevent the genetically modified organism from contaminating the wild populations for better biosafety. The researchers engineered an Escherichia coli to incorporate synthetic amino acids into essential proteins by using the freed "stop" codon. When the bacteria are not fed the synthetic amino acids, they cannot produce their essential proteins will die. This strategy yields bacteria that are very unlikely to survive in the wild environments lacking the synthetic amino acid.
After freeing the "TGA" stop codons, the researchers are aiming an even more ambitious projects. One of them is to create a 57-codon E. coli genome. They want replace codons AGA, AGG, AGC, AGU, UUA, UUG, and UAG by synonymous alternatives. In total, the recoded genome design had 62,214 codon replacements across 3,548 genes.
The team pursued this by a different approach from the MAGE and CAGE method. They divided the recoded genome into 87 segments, each about 50 kb long. The researchers planned to synthesize these 87 segments from scratch and then integrating each segment into a separate strain of E. coli. Till now, they assembled and tested only two thirds of the replacement and is still far from achieving a fully operational 57-codon E. coli.
In the recently launched GP-write project, scientists are aiming to recode the entire human genome. The story of recoding will undoubtedly be continued.
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