Streamlined cell-free protein synthesis using wheat germ extract.

Wheat germ extract (WGE)-based cell‑free translation is a robust and versatile platform for small‑scale, high‑throughput production of diverse eukaryotic proteins. Although the system has been optimised for efficient protein synthesis using cap‑independent translation‑compatible vectors and bilayer in vitro translation formats, it has traditionally relied on separate transcription and translation steps. To streamline this workflow, a bilayer, coupled in vitro transcription-translation (cIVTT) protocol was developed in which both processes are integrated into a single reaction. After optimisation of the reaction mixture composition and reaction conditions, protein yields comparable to those obtained with the conventional two‑step bilayer in vitro translation method were achieved. Notably, the cIVTT protocol remained effective at reduced temperatures, thereby supporting the enhanced production of human cardiac muscle Troponin I (cTnI), a protein with demanding folding requirements. This simplified and time‑efficient approach enables microgram‑scale protein production and is well suited for high‑throughput applications. KEY POINTS: • Streamlined WGE cell-free protein synthesis workflow • No plasmid linearisation needed before coupled in vitro transcription/translation • Soluble eukaryotic protein yields on par with two‑step bilayer format.