New way to make fusion proteins

July 10, 2012 § Leave a comment

The new way of making fusion proteins overcomes some of the challenges of the traditional genetic approach. Image provided by Hidde Ploegh.

Biochemists and molecular biologists devote quite some time to making protein-protein fusions, such as tagging their favorite protein with green fluorescent protein or glutathione-S-transferase. But what if the conventional fusion process stifles the activity of one of the proteins? In a paper out this week in the Proceedings of the National Academy of Sciences, a group of researchers report a new way to make  protein fusions that overcomes this and other problems.

The standard way of making protein fusions is to use genetics. Researchers stick the open reading frame of one protein next to the open reading frame of another protein and then get cells to turn this artificial genetic construct into protein.

However, this approach doesn’t always work well. One problem is that the proteins sometimes don’t fold correctly and thus don’t function properly. Another limitation, explains the lead author of the PNAS paper, Hidde Ploegh at the Whitehead Institute, is that the termini of many proteins, including antibodies and chemokines, are important for biological activity. “When two proteins that both require their N- or C-termini for activity are fused genetically, it is fair to reason that the activity of at least one of the fusion partners is impaired,” he says.

So Ploegh and his team decided to develop a method to make “bispecifics,” proteins that have two different functional ends. The method involves a transacylation reaction catalyzed by a bacterial enzyme called sortase A to put on a small reactive moiety at the N- or C-terminus of one of the proteins to be fused. The other protein has another reactive moiety on its corresponding N- or C-terminus.

Under the correct conditions, the two reactive moieties snap together by a process called click chemistry. The result? Two proteins that are attached head to head or tail to tail, leaving their biologically active ends free to do their jobs.

“The strategy is straightforward and broadly applicable,” says Ploegh. “Labs that have access to standard protein expression will be able to prepare bispecifics using this method.”

Bispecifics aren’t merely laboratory toys for biochemists and molecular biologists. As Ploegh and colleagues point out in their paper, the protein fusions are being investigated as treatment options for cancer, autoimmune diseases and brain disorders.

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