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Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.

Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo