In Vitro Protein Synthesis in Semipermeable Artificial Cells

Area of Science:

• Biotechnology
• Synthetic Biology
• Biochemistry

Background:

• Conventional cell-free protein synthesis (CFPS) is costly due to lysate preparation and purification.
• Incorporating noncanonical amino acids (ncAAs) into proteins requires specialized enzymes and methods.
• Marginally stable proteins pose challenges for in vitro expression systems.

Purpose of the Study:

• To develop a novel, cost-effective CFPS system using layer-by-layer (LbL) polymer assembly.
• To create an encapsulated cell system (eCells) for improved in vitro protein synthesis.
• To demonstrate the efficient incorporation of ncAAs and stabilization of proteins within eCells.

Main Methods:

• Developed an encapsulated cell system (eCells) using LbL polymer assembly.
• Performed in vitro CFPS within eCells, including the incorporation of ncAAs like photocaged-cysteine (PCC) and Boc-lysine.
• Assessed protein yield, cost-effectiveness, scalability, and protein stabilization.
• Utilized fluorescent activated cell sorting (FACS) to analyze lysate partitioning.

Main Results:

• eCells achieved 55% of the productivity of standard dialysis CFPS at only 13% of the cost.
• Demonstrated cost-effective incorporation of ncAAs into Peptidyl-prolyl cis-trans isomerase B (PpiB).
• Showcased the stabilizing effect of the eCell crowding environment on marginally stable proteins like pyrrolysl tRNA synthetase (PylRS).
• Confirmed successful lysate encapsulation within eCells using FACS.

Conclusions:

• eCells represent a scalable and cost-effective platform for in vitro protein synthesis.
• This system facilitates the economical incorporation of ncAAs and enhances the stability of challenging proteins.
• eCells offer a promising alternative to traditional CFPS methods for various biotechnological applications.