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Princeton Team Creates Functional New Synthetic Proteins

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – In a paper appearing online this week in PLoS ONE, Princeton University researchers reported that they have successfully created synthetic proteins that can function in Escherichia coli.

Using a so-called binary code method that relies on strategic placement of polar and non-polar residues, the team made more than a million stably folded strings of amino acids from genetic sequences distinct from those known to occur naturally. They then screened these synthetic proteins in dozens of E. coli strains missing essential genes, identifying artificial proteins that could substitute for the bug's own proteins.

"What we have here are molecular machines that function quite well within a living organism even though they were designed from scratch and expressed from artificial genes," senior author Michael Hecht, a Princeton chemistry researcher, said in a statement. "Our work suggests that the construction of artificial genomes capable of sustaining cell life may be within reach."

Using the binary code approach, Hecht and his team designed genes coding for 1.5 million new amino acid sequences, each capable of folding into stable, 102 residue, 4-helix bundle structures.

They then screened the functionality of these newly created proteins by inserting their sequence library into a set of 27 E. coli knockout strains missing genes known to be necessary for E. coli survival. The researchers found that 18 of the artificial proteins could rescue the growth of four E. coli mutant strains missing the essential enzyme-coding genes serB, gltA, ilvA, or fes.

"[T]he information encoded in these artificial genes is completely novel," lead author Michael Fisher, a University of California at Berkeley post-doctoral researcher who participated in the study as a graduate student at Princeton, said in a statement, "it does not come from, nor is it significantly related to, information encoded by natural genes, and yet the end result is a living, functional microbe."

Moreover, when they removed all four of these naturally occurring genes at once in a single strain, the researchers showed that the bacterial cells could survive in the absence of these genes when they co-expressed artificial proteins identified in the screen.

"The novel proteins are substantially less active, and may function by different mechanisms than the natural proteins they replace," they explained. "Nonetheless, co-expression of several novel proteins rescues a strain in which multiple natural genes were deleted simultaneously."

Given their findings so far, the researchers speculated that it should be possible to find even more functional synthetic proteins by expanding both the sequence library and deletion strain set used to screen these new sequences.

"The results described here suggest that the toolkit for synthetic biology need not be limited to genes and proteins that already exist in nature," they concluded, arguing that their ability to substitute synthetic proteins for natural proteins in E. coli represents "an initial step toward the construction of artificial genomes."