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Cell-free protein expression—A “first-aid kit” for challenging protein expression
Release date:
2024-11-06
Most efforts in biosynthesis have focused on living cells, yet the cellular machinery and intracellular pathways inevitably pose significant challenges. Primarily due to the complexity of living-cell life systems—along with the irreversibility of cell growth, intrinsic intracellular noise, and the barrier imposed by the cell membrane—these factors severely constrain the engineering of biological components.
Furthermore, the intracellular environment of heterologous host cells differs significantly from the native milieu; these dissimilarities or mismatches can give rise to phenomena such as inclusion body formation, protein misfolding, metabolic burden imbalance, and cytotoxicity toward the host cell.
In the face of the complex characteristics and challenges of cellular systems, a new field of cell-free synthetic biology is emerging. Compared with protein expression in cellular systems, cell-free protein expression platforms offer distinct advantages.
CFPS Case Study — Inclusion Body Protein
Inclusion bodies are membrane‑enclosed, high‑density, insoluble protein aggregates that form when heterologous genes are expressed in prokaryotic cells. In cell‑free protein expression systems, the addition of molecular chaperones and other components that assist protein folding can convert some proteins that would otherwise be expressed as inclusion bodies in Escherichia coli into soluble forms that remain in the supernatant.
IL‑2 is an important cytokine belonging to the chemokine family; however, in prokaryotic expression systems, IL‑2 is often produced as inclusion bodies. We evaluated the soluble expression levels and assessed the biological activity of IL‑2 using cell-free expression with PLD, expression in Brand A (Overseas) CHO cells, and prokaryotic expression with Brand B (China).
Figure A: IL‑2 is expressed in soluble form in the cell‑free supernatant of PLD, with a distinct band visible. Figure B: Cell‑based activity assays demonstrate that the IL‑2 produced via the cell‑free expression system of PLD exhibits activity comparable to that of Brand A (Overseas) produced in CHO cells, and superior to that of the prokaryotic‑expressed product from Brand B (China).
CFPS Case Study — Disulfide-Bonded Proteins
In protein structure, disulfide bonds are crucial for maintaining conformational stability. For example, in antibody molecules, the heavy and light chains are linked by disulfide bonds, which help preserve the correct conformation necessary for recognizing specific antigens. If these disulfide bonds misfold or are lost, the protein may become inactivated or degraded. Cell-free systems aim to simultaneously carry out protein synthesis, folding, and disulfide bond formation within the same spatial environment.
A protein containing six disulfide bonds was analyzed for its soluble expression profile using both a cell-free PLD-based expression system and a cell-based expression system; concurrently, the activity of the cell-free PLD‑derived protein was compared with that of a commercially available product.
Figure A: Protein structure, with the yellow regions corresponding to disulfide bonds.
Figure B: The protein was expressed in soluble form in the cell-free supernatant of PLD, yielding a distinct band. No band was detected in the supernatant from the cell-based expression system.
Figure C: PLD produces this protein with activity comparable to that of a commercially available product.
CFPS Case Study — Toxic Protein
Restriction endonucleases pose a significant challenge to the recombinant expression of proteins in cells. When recombinant proteins are overexpressed in heterologous hosts, their DNA‑cleaving activity severely impairs host growth and the expression of the target protein, making it difficult to obtain biologically active product.
Using PLD cell-free rapid expression technology, the BamHI restriction enzyme was successfully expressed. The BamHI restriction enzyme produced by PLD cell-free protein expression was incorporated into plasmids at concentrations of 0, 0.001, 0.0015, 0.015, and 0.15 µg/µL, and subjected to digestion at 37°C for 1 hour to assess enzymatic activity.
Figure A: The BamHI restriction enzyme was expressed in a cell-free PLD system; after crude purification, gel electrophoresis revealed the expected band. Figure B: Plasmids corresponding to lanes 2, 3, 4, and 5 were digested into linear templates, demonstrating that the BamHI restriction enzyme produced by cell-free PLD protein expression is enzymatically active, with detectable cleavage activity at a minimum concentration of 0.001 µg/µL.
Perotin Cell-Free Protein Expression Service – Challenging Protein Expression
Perotin Bio leverages its proprietary cell-free protein expression platform, enabling direct in vitro synthesis of proteins without relying on living cells. The company has successfully achieved efficient and rapid production of proteins that are difficult to express within cells, effectively addressing the challenges of such proteins. Perotin’s CFPS system boasts a success rate exceeding 96% and can produce toxic proteins, inclusion-body proteins, disulfide‑bonded proteins, and more.
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