Researchers Overcome Major Protein Engineering Hurdle

AGENTEX Platform and Cell-Free Production

For more than two decades, scientists tried to instruct biological systems like E. coli to produce non-natural proteins by reprogramming their DNA. That process proved exceptionally difficult, time-consuming, and yielded feeble results, according to the Harvard team. To bypass modifying genomes or working with living cells, researchers at Harvard Medical School and the Wyss Institute for Biologically Inspired Engineering—specifically within the lab of geneticist George Church—developed AGENTEX. Felix Radford, first author and an HMS research fellow in genetics based in the Church Lab, noted that this platform lets scientists synthesize brand-new genetic codes on demand inside test tubes, scaling them up to manufacture proteins that far surpass natural evolutionary limits. The system relies on custom-engineered transfer RNAs (tRNAs) and ribosomes added to standard lab lysate solutions, which churn out new proteins without interfering with natural cellular machinery.

Expanding the Amino Acid Alphabet

Natural proteins are built from combinations of just 20 amino acids, each coded by a triplet of DNA or RNA bases called a codon. Because there are roughly three times as many codons as amino acids, many codons exhibit redundancy—such as UCU, UCC, UCA, and UCG all coding for serine—suggesting they could be reprogrammed. After nine years of effort, the Church Lab freed up one codon in E. coli in 2013, and it took another decade to free up a second codon, allowing cells to make proteins containing up to 22 amino acids. Global progress remained slow because codon reprogramming hampers normal cell functions, and engineered organisms require strict containment. Traditional approaches demanded roughly ten years for each additional amino acid introduced, whereas AGENTEX introduces the capacity for 34 simultaneously while causing virtually zero genomic damage, as noted by Church, who serves as the Robert Winthrop Professor of Genetics in the Blavatnik Institute at HMS and leads synthetic biology as founding core faculty at the Wyss Institute.

Revising Transfer RNA Dogma

The breakthrough builds on a discovery regarding transfer RNAs (tRNAs), which add amino acids into a growing protein chain. Every tRNA features the genetic sequence CCA on its tail end. Decades of scientific dogma held that any alternative sequence flags the tRNA as defective, causing enzymes to deny it an amino acid cargo and ribosomes to bar it from delivery. Radford and colleagues discovered that enzymes actually allow some tRNAs with alternative tail-end sequences to receive amino acids. Using a tool named tSCAN built as part of AGENTEX, the team identified functional nonstandard sequences, including CGA. Because non-CCA tRNAs are barred from natural ribosomes, scientists can engineer ribosomes that exclusively work with specific alternative sequences like CGA, pairing them with custom amino acids.

Automated Workflow and Future Applications

AGENTEX—short for automated genetic tRNA expansion—provides an end-to-end workflow utilizing free software written by Church’s team that runs on an open-source robot sold by Opentrons. Researchers use AGENTEX to design and produce tRNAs with different non-CCA end sequences and up to 34 amino acids, batch-testing them in lysate solutions to identify successful variations. The platform then designs matching ribosomes and combines everything in lysate solutions to synthesize the target proteins. Beyond protein production, tSCAN offers a new method to study tRNAs in general, including mutations linked to diseases like diabetes and hearing loss. The research team also envisions integrating artificial intelligence into AGENTEX to further optimize protein design for developing therapeutics, materials, and food.

Frequently Asked Questions

What is AGENTEX?

AGENTEX is a cell-free synthetic protein production tool developed at Harvard that allows researchers to design and produce proteins using up to 34 custom amino acids without altering living genomes.

How does AGENTEX differ from older protein engineering methods?

Older methods required reprogramming an organism’s DNA over decades—averaging about ten years per added amino acid—whereas AGENTEX operates in test tubes using lysate solutions and custom tRNAs and ribosomes, making 34 codons customizable at once.

What was the key biological discovery behind AGENTEX?

Researchers discovered that transfer RNAs (tRNAs) do not strictly require the traditional CCA tail-end sequence to receive amino acid cargo from enzymes, overturning decades of scientific dogma and enabling side-by-side protein processing.

Who funded this research?

Financial backing for the project was provided by the Department of Energy, the National Science Foundation, and an HMS Dean’s Innovation Award for the Use of Artificial Intelligence in Education, Research, and Administration.