Cell-free expression with the toxic amino acid canavanine

Canavanine is a naturally occurring noncanonical amino acid, which is analogous to arginine. It is a potent antimetabolite and natural allelochemic agent, capable of affecting or blocking regulatory and catalytic reactions that involve arginine. Incorporated into proteins at arginine positions, canavanine can be detrimental to protein stability and functional integrity. Although incorporation of canavanine into proteins has long been documented, due to its toxicity, expression in Escherichia coli and other common hosts remains a considerable challenge. Here, we present a simple, cell-free expression system with markedly improved performance compared to heterologous expression. The cell-free expression system does not require any tuning besides substitution of arginine by canavanine. We show that our technique enables highly efficient protein expression in small volumes with arginine being fully replaced by canavanine for functional and structural studies.