Technical Support
Deeply interpret the latest scientific research progress and application cases in the fields of cell-free protein expression and membrane protein preparation.
What is cell-free protein expression? What are its technical principles?
Cell-free protein synthesis refers to a biosynthetic process in which the molecular machinery required for intracellular protein synthesis—such as RNA polymerase, ribosomes, and transcription‑translation cofactors—is isolated and supplemented with nucleotides, amino acids, energy substrates, and a genetic template, enabling the direct in vitro production of proteins without the involvement of living cells. The underlying principle of cell-free protein synthesis is the central dogma, with transcription and translation carried out outside the cell. Specifically, within a cell‑free reaction system, the DNA template encoding the target protein is transcribed into mRNA by RNA polymerase; subsequently, using aminoacyl‑tRNA synthetases, ribosomes, and tRNAs, the mRNA serves as a template for translating the desired protein.
What are the advantages of cell-free protein expression compared to conventional cellular expression? And in which fields is it particularly well suited?
First, the reaction cycle is short; the absence of cellular protein expression eliminates the need for conventional steps such as cell transfection and cultivation, enabling protein expression within a few hours to one day. With… Suzhou Perotin (PLD technology) Take the cell-free kit as an example—proteins can be produced in as little as one hour, making it ideal for rapid protein delivery.
Second, it can be integrated with automated equipment to enable high-throughput protein expression on microtiter plates—such as 96-well plates—meeting the demand for rapid, large-scale protein screening. This makes it well suited for applications like directed enzyme evolution and AI-driven protein design.
Third, the system supports a wide variety of recombinant proteins. While some proteins exhibit low yields or fail to be expressed in conventional cell-based expression systems, cell-free systems can circumvent the intrinsic physiological constraints of cells, making them well suited for applications such as the production of cytokines, antibodies, membrane proteins, toxic proteins, and inclusion-body proteins.
Fourth, it offers high degrees of freedom and controllability, enabling the incorporation of non‑natural amino acids and site‑specific modifications, making it well suited for ADC drug development. Moreover, cell‑free protein expression is shorter in duration and simpler to perform compared with conventional cell‑based expression workflows.
What challenges can cell-free protein expression help address? I’ve been struggling to express a membrane protein—could cell-free systems enable its production?
Cell-free protein expression offers an ideal solution to the longstanding challenges of conventional protein‑expression workflows—namely, their lengthy timelines and low efficiency—particularly for difficult-to-express targets such as membrane proteins and toxic proteins. Cell‑free systems can successfully produce membrane proteins; in China, Perotin Bio provides a cell‑free protein‑expression kit specifically optimized for membrane proteins. By simply following the protocol—adding the DNA template of the target membrane protein along with an appropriate surfactant or nanodiscs to the reaction mixture—membrane proteins can be obtained within a few hours to one day.
Will the activity of a protein expressed in a cell-free system be affected, and is purification necessary?
The cell-free expression system for Perotin can facilitate proper protein folding by optimizing reaction temperature, incorporating molecular chaperones, and adding cofactors, yielding proteins with activity comparable to, or even superior to, that achieved in cellular expression systems. As with conventional cell-based expression, the products of cell-free systems contain residual proteins in addition to the target protein. For assays of antibody affinity and enzymatic activity, cell-free‑expressed proteins can be tested directly without purification; for applications requiring high protein purity or involving cell‑based experiments, standard purification protocols used for conventional protein expression may be followed.
Can cell-free systems express disulfide-bonded proteins? Are there any post-translational modifications?
Cell-free systems can express disulfide‑bonded proteins, though this varies depending on the manufacturer’s product. All cell‑free protein expression products offered by Perotin Biotech support the production of disulfide‑bonded proteins. Cell‑free systems derived from prokaryotes lack glycosylation, whereas those derived from eukaryotes exhibit limited glycosylation.
What is the efficiency of the cell-free system? To what scale can it be scaled up?
The efficiency of cell-free systems typically depends on the target protein, with expression levels ranging from μg/mL to several hundred μg/mL. For example, using the Perotin Bio cell-free protein expression kit, protein yields can reach up to 3 mg/mL. Cell-free systems are scalable, and large-scale production at the hundred‑liter scale has already been reported.
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