Proteins are stubborn things, and achieving high yields of properly folded and modified polypeptides can become a truly Herculean task, with cell-based production often hampered by issues of efficiency and toxicity as well as the difficulty of collecting target protein.

Existing cell-free lysate-based systems offer a promising alternative but not a perfect solution. “You can do cell-free preparation in Escherichia coli on the multiliter scale,” says Kirill Alexandrov of the University of Queensland, Australia. “But if you go to the eukaryotic world, which you have to do because E. coli doesn't properly fold complex eukaryotic proteins, then things become much more difficult.” Preferred systems like rabbit reticulocyte lysate and wheat germ extract fall short when it comes to yield and scalability, and Alexandrov was keen to develop a more efficient and universal approach to eukaryotic protein production.

The capacity to specifically and efficiently engage lysate translational machinery with exogenous mRNAs is vital for successful cell-free protein production, and Alexandrov's team achieved a breakthrough in this regard by reexamining the leader sequences on their starting transcripts. Previous evidence has suggested that relatively unstructured poly(A)-rich sequences stimulate translational complex assembly, and by designing a customized 5′ untranslated region based on this concept, they considerably boosted the efficiency of translation for linked transcripts in virtually any cell-free system. “This translation is truly species-independent and worked in all organisms that we tested, including both eukaryotes and prokaryotes,” says Alexandrov.

His team has worked closely with one particularly intriguing organism, the trypanosome Leishmania tarentolae, with characteristics that make it a promising candidate for cell-free extracts. “It's essentially a transitional organism between prokaryotes and eukaryotes,” he says, “because they have prokaryotic gene expression [machinery] but eukaryotic protein folding and modification machinery.” Another potent advantage of this organism is the existence of a ubiquitous 35-nucleotide leader sequence on all endogenous mRNAs; as a result, translation of these could be readily blocked with a Leishmania-specific antisense nucleotide, eliminating the need for RNase digestion as in other extract-based methods.

This combination of L. tarentolae extract and their species-independent translational sequence proved potent, resulting in markedly higher yield of protein than competing eukaryotic extracts—up to 300 micrograms of protein per milliliter in 90 minutes—with a diverse array of protein-coding sequences, although the efficiency of folding varied depending on the protein in question. As a further demonstration of the utility of their method, the researchers monitored diffusion and interaction behavior of individual proteins directly in the cell-free translation mixture using fluorescence correlation spectroscopy.

Based on these initial experiments, Alexandrov is encouraged that his method offers a strong combination of efficiency and the capacity to ratchet up eukaryotic protein production to the liter scale at a fraction of the cost of existing commercial systems—a serious boon to scientists looking to generate crystals or produce antibodies. In contrast, there are also important advantages in 'going small', and the capacity for highly multiplexed protein production using the species-independent translational sequence leader, either with L. tarentolae extract or any other extract system, could enable powerful, high-throughput interaction analysis studies or even new approaches for tackling proteomic research. “You can find an organism, make an extract and back-translate its genome,” says Alexandrov. “Craig Venter estimates a total pool of 20 million genes on the planet, and most of them are in microorganisms on which we have no handle—and so this could be a way of quickly generating expressed proteomes.”