Reagenti e strumenti per immunologia, biologia cellulare e biologia molecolare

Cell-Free Protein Expression

In this approach, a solution containing all the essential cellular components required for protein synthesis (e.g., ribosomes, tRNAs, enzymes, cofactors, amino acids, etc.) is used to transcribe and translate an input nucleic acid template, such as plasmid DNA, linear DNA, or mRNA. CFPS allows for the rapid generation of desired proteins within just a few hours, in contrast to traditional in vivo protein expression methods, which can take several days or longer.

CFPS is highly versatile and supports a wide range of applications. Its speed and simplicity enable high-throughput protein expression, making it ideal for tasks such as protein engineering, mutagenesis studies, and enzyme screening. Furthermore, CFPS is commonly employed to produce proteins for biophysical studies and structure-function analyses. Additional applications include the production of proteins that are toxic to living host cells, the expression of proteins incorporating modified or unnatural amino acids, and the addition of post-translational modifications in certain CFPS systems. Thanks to their efficiency, scalability, and cost-effectiveness, CFPS systems are also widely utilized in metabolic engineering and synthetic biology.

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Overview of CFPS Process

Figure: General workflow for preparation of cell-free extract and set up of CFPS reactions. A visualization from cell growth to the CFPS reaction is depicted above for a new user, highlighting the main steps involved.(1)

Cell-Free systems are advantageous for producing complex proteins

Simplify your process with no need for cell cultivation, no more toxicity during cultivation! This open system allows addition of additives, thus creating an environment favorable to protein folding.
Rapid Production: Get your proteins quickly with fast synthesis times (1 week for large-scale production)!
High-throughput productions available!
Efficiency: Directly use DNA templates for efficient protein production. A powerful technology to make proteins that were previously difficult to produce.
Adapted for proteins like: Membrane proteins, cytotoxic proteins, complex folding proteins, viral proteins antigen for antibody generation and more broadly proteins which are difficult to express in classic systems.

What are the advantages of cell-free protein synthesis (CFPS) systems compared to cell-based systems?

CFPS is cost-efficient, as it eliminates the need for specialized facilities for bacterial or cell cultures. It is also fast, completing protein synthesis in just 3 hours, and offers easy control. Additionally, CFPS systems allow for real-time monitoring and modification of protein synthesis reactions.
As an open system, CFPS enables precise control of reaction conditions and scalability of production. For example, adding metal ions (such as Fe²⁺, Mn²⁺, and Cu²⁺) can enhance the accurate synthesis of proteins. CFPS systems can also serve as a platform for producing toxins, such as antimicrobial peptides.
The open nature of CFPS is particularly beneficial for prototyping new metabolic pathways and genetic circuits, providing controllable chemical enzymatic reactions among active substances.

Can eukaryotic proteins be expressed using CFPS systems?

Yes, CFPS is well-suited for eukaryotic protein expression. As an enzymatic cascade system with open characteristics, CFPS allows the addition of specific components required for eukaryotic protein synthesis. Some target proteins, such as antibody fragments (scFv and VHH), do not rely on post-translational modifications for functionality. When expressed using CFPS and HEK293 systems, there is no significant difference in their functional activity.

Is CFPS suitable for high-throughput production, and what are its yields? Can it detect low-abundance proteins?

CFPS is highly suitable for high-throughput protein production, with yields reaching up to the milligrams-per-milliliter (mg/mL) range. Antibodies and antibody fragments are typically produced at levels of around 100 µg/mL. For low-abundance proteins, specific detection methods need to be developed. If more details are provided, we can assist with designing a tailored detection methodology.

Does CFPS offer advantages for proteins that are challenging to express in prokaryotic systems?

Yes, CFPS is particularly advantageous for expressing proteins that are difficult to synthesize in prokaryotic systems. Its open nature allows the inclusion of additional components, such as eukaryotic lysates, to support accurate synthesis of eukaryotic proteins.