Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Cell-free protein synthesis (CFPS) enables rapid protein production from DNA.
• Genetic code expansion allows site-specific incorporation of unnatural amino acids (UAAs) using orthogonal translation systems (OTS).
• Existing OTS components (orthogonal tRNA and aminoacyl-tRNA synthetase - aaRS) are often unstable and difficult to prepare, limiting CFPS applications.

Purpose of the Study:

• To develop a more stable and efficient method for UAA incorporation in cell-free protein synthesis.
• To reduce the complexity, effort, and time required for expressing UAA-containing proteins.
• To enable the use of previously challenging reagents like pyrrolysyl-tRNA synthetase in cell-free systems.

Main Methods:

• Development of an endogenously introduced orthogonal pair for cell-free systems.
• Utilizing pyrrolysyl-tRNA synthetase within the cell-free reaction.
• Optimization of reaction conditions for stability and efficiency.

Main Results:

• Significantly reduced complexity, effort, and time for UAA-containing protein expression.
• Increased stability and maximal suppression efficiency of the orthogonal translation system.
• Successful integration of pyrrolysyl-tRNA synthetase into the cell-free system.

Conclusions:

• The developed system offers an improved and accessible platform for UAA incorporation into proteins.
• This advancement expands the genetic repertoire usable in vitro, opening new avenues for bioengineering.
• The enhanced stability and efficiency make UAA incorporation more practical and widely applicable.