Well begun isn’t always half done. Sprinters can stumble after they’ve taken a few strides out of the starting blocks. Car engines can stall after they’ve turned over a few times. And recombinant proteins can stop elongating after the first few amino acids have been translated from mRNA.

This last snag is the least obvious to most people, but it matters a great deal to manufacturers of therapeutic proteins, who are always looking for ways to enhance the efficiency of the genetically engineered host cells that churn out protein-based drugs, vaccines, and diagnostics.

Manufacturers of therapeutic proteins, then, try to introduce efficiencies at the rate-limiting steps in protein synthesis. The rate-limiting step known as translation initiation has already attracted a lot of attention. But soon after translation initiation there is another rate-limiting step that may prove even more important. It is called early elongation.

Early elongation is the subject of a new study by scientists at Washington University School of Medicine in St. Louis. According to these scientists, the efficiency of protein synthesis strongly depends on the nucleotide sequence positions 7–15, which correspond to amino acid positions 3–5. That is, when a stubby protein begins to grow, amino acid by amino acid, elongation often stalls at the third, fourth, or fifth amino acid. The scientists also determined that the stalling effect’s impact on protein yield is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure.

Detailed findings appeared December 18 in Nature Communications, in an article titled, “A short translational ramp determines the efficiency of protein synthesis.”

“To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays,” the article’s authors wrote. “Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions.”

The Washington University scientists, led by Sergej Djuranovic, PhD, assistant professor, cell biology and physiology, stumbled on the importance of the first few amino acids when an experiment for a different study failed to work as expected. The researchers were looking for ways to control the amount of protein produced from a specific gene.

“We changed the sequence of the first few amino acids, and we thought that the change would have no effect on protein expression, but instead, the change increased protein expression by 300%,” Djuranovic said. “So, then we started digging into why that happened.”

The researchers turned to green fluorescent protein, a tool used in biomedical research to estimate the amount of protein in a sample by measuring the amount of fluorescent light produced.

“To determine the role of amino acid sequence, we created a library of an otherwise codon-optimized eGFP gene with insertion of nine random nucleotides after the second codon,” the article’s authors detailed. “Sequencing of the plasmid library revealed 259,134 unique sequences out of the 262,144 possible synthetic eGFP constructs. These were identical except for the 3rd–5th codons (nucleotides 7–15) of the open reading frame. These three codons code for 9,261 different tripeptides including truncated peptides due to the presence of one or more stop codons.”

The brilliance of the different versions of green fluorescent protein varied a thousandfold from the dimmest to the brightest, the researchers found, indicating a thousandfold difference in the amount of protein produced.

After further experimentation, Djuranovic and colleagues identified certain combinations of amino acids at the third, fourth, and fifth positions in the protein chain that gave rise to sky-high amounts of protein. Moreover, the scientists determined that the same amino acid triplets not only ramped up production of green fluorescent protein, which originally comes from jellyfish, but also production of proteins from distantly related species like coral and humans.

“There are so many ways we could benefit from ramping up protein production,” Djuranovic declared. “If you can make each bacterium produce 10 times as much protein, you only need 1/10th the volume of bacteria to get the job done, which would cut costs tremendously. This technique works with all kinds of proteins because it’s a basic feature of the universal protein-synthesizing machinery.”

“In the biomedical space, there are many proteins used in drugs, vaccines, diagnostics, and biomaterials for medical devices that might become less expensive if we could improve production,” he continued. “And that’s not to mention proteins produced for use in the food industry—there’s one called chymosin that is very important in cheese-making, for example—the chemical industry, bioenergy, scientific research, and others. Optimizing protein production could have a broad range of commercial benefits.”

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