——— Jenny Wang

Q1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell free expression is more beneficial than cell production.

Figure 1. The CFPS platform is amenable to direct manipulation of the environment of protein production because it is an open system. (Silverman, Andrew D., Nadanai Laohakunakorn, and Michael C. Jewett.)

Two Cases Where CFPS Outperforms In Vivo Production:

1. Toxic Protein Production
   • Example: Antimicrobial peptides (AMPs) or pore-forming proteins that kill host cells during in vivo expression.
   • CFPS Benefit: Toxicity is irrelevant in a cell-free system, enabling high-yield synthesis without cell death or metabolic interference.
2. Non-Canonical Amino Acid (ncAA) Incorporation
   • Example: Site-specific incorporation of fluorescent tags (e.g., BODIPY) or click-chemistry handles (e.g., azidohomoalanine) for protein labeling.
   • CFPS Benefit: Simplified addition of modified tRNAs and amino acids, avoiding the need to engineer orthogonal translation systems into living cells.

Q2. Describe the main components of a cell-free expression system and explain the role of each component.

Cell Extract (Crude Lysate or Reconstituted System)

• Source: Typically from E. coli, wheat germ, rabbit reticulocyte, or insect cells.
• Function: Contains the core transcription and translation machinery, including:
  • RNA polymerases
  • Ribosomes
  • tRNAs
  • Aminoacyl-tRNA synthetases
  • Translation factors
  • Energy-regenerating enzymes
• Role: Carries out transcription (DNA → mRNA) and translation (mRNA → protein).

DNA Template