- 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.
Cell-free protein synthesis (CFPS) leverages cell lysates (e.g., E. coli, wheat germ, mammalian extracts) to produce proteins without intact cells, offering unparalleled flexibility and control over experimental conditions. Here’s how it outperforms traditional in vivo methods:
1. Flexibility in Experimental Design
- Open System: No cell walls or membranes restrict access, enabling:
- Direct manipulation of reaction conditions (pH, redox, temperature).
- Real-time monitoring and adjustments (e.g., adding nucleotides, cofactors).
- Customizable Energy Sources: ATP regeneration systems (e.g., creatine kinase) can be optimized for prolonged reactions.
2. Precise Control Over Variables
- Tunable Expression: Adjust DNA/RNA templates, ribosome concentrations, or translation machinery on-demand.
- Reduced Complexity: Eliminates cellular processes (e.g., division, metabolism) that complicate in vivo systems.
- Rapid Prototyping: Reactions complete in hours (vs. days for cloning/transformation in vivo).
Cases Where CFPS Outperforms Cell-Based Production
1. Toxic or Membrane Protein Production
- Problem: Many proteins (e.g., ion channels, antimicrobial peptides) kill host cells during in vivo expression.
- CFPS Solution: Lysates lack viability constraints, enabling high-yield synthesis of toxic proteins.
- Example: Production of voltage-gated sodium channels (toxic to E. coli) for structural studies.
2. Non-Natural or Modified Proteins
- Problem: Incorporation of non-canonical amino acids (ncAAs) or post-translational modifications (PTMs) is inefficient in vivo.
- CFPS Solution:
- Direct addition of ncAAs (e.g., p-azidophenylalanine) to the lysate.
- Use of specialized extracts (e.g., insect cells for glycosylation).
- Example: Synthesis of antibody-drug conjugates with site-specific ncAA incorporation.