Nebulabio provide a cutting-edge, high-throughput Cell-Free Protein Synthesis (CFPS) service, enabling the rapid production of functional proteins without the use of living cells. This platform bypasses the time-consuming and often cumbersome steps of cloning, transformation, and cell culture associated with traditional in vivo expression systems. Our service is designed for researchers and developers who require speed, flexibility, and the ability to express challenging proteins that are toxic, insoluble, or require non-natural components. By leveraging optimized E. coli, wheat germ, or mammalian cell lysates, we offer a versatile solution for producing proteins for functional studies, high-throughput screening, antibody generation, and early-stage therapeutic candidate evaluation. From DNA template to purified protein in a matter of hours to days, we deliver a robust and scalable alternative to accelerate your research and development pipeline.
☞Introduction to Cell-Free In Vitro Protein Expression
Cell-free protein expression is a methodology where protein synthesis is reconstituted in vitro using the essential cellular machinery extracted from cells. The core system consists of a lysate containing ribosomes, translation factors, tRNAs, and enzymes, supplemented with amino acids, energy sources (ATP, GTP), and other necessary cofactors. When a DNA template (linear or circular) or an mRNA encoding the target protein is added to this reaction mix, the machinery reads the genetic code and synthesizes the protein.
Figure1.The various advantages of cell-free synthesis of membrane proteins is described.
Key Advantages of Our Cell-Free Platform:
● Unmatched Speed: Produce microgram to milligram quantities of protein in a few hours to one day, drastically faster than cell-based systems (which require days to weeks for culture growth).
● Expression of Challenging Proteins: Ideal for producing proteins that are toxic to host cells, insoluble in cellular environments, or contain non-natural amino acids (nnAAs), toxic moieties, or radioactive labels.
● High Throughput & Flexibility: Easily parallelize hundreds of reactions in microplates to screen different constructs, expression conditions, or variants simultaneously. The open reaction environment allows direct control over redox conditions, chaperones, and detergents to favor soluble folding.
● Simplified Purification: Reactions lack the complex background of host cell proteins, often leading to simpler purification schemes. Proteins can be synthesized with affinity tags (His, GST, Strep) for direct capture from the reaction mix.
● On-Demand & Scalable: Produce protein exactly when needed, minimizing waste. The system is linearly scalable from a 50 µL micro-reaction to milliliter-scale batch or continuous-exchange formats for higher yields.
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☞Applications of Cell-Free In Vitro Protein Expression
This technology is transformative across multiple disciplines:
● Rapid Protein Production for Functional Analysis: Quickly express and test mutant protein libraries, domain fragments, or novel constructs to map structure-function relationships. ● High-Throughput Screening & Drug Discovery: Generate targets (enzymes, receptors) or potential biotherapeutics (antibody fragments, peptides) directly in microtiter plates for immediate use in activity assays or binding screens. ● Protein Engineering & Evolution: Couple expression directly to selection pressures (e.g., ribosome display, mRNA display) for in vitro evolution of proteins with enhanced stability, affinity, or activity. ● Production of Difficult Proteins: Express membrane proteins in the presence of detergents or nanodiscs, cytotoxic proteins (e.g., nucleases, apoptosis inducers), and proteins with site-specific nnAA incorporations for click chemistry or probes. ● Synthetic Biology & Prototyping: A foundational tool for building synthetic biological systems and rapidly prototyping genetic circuits by expressing their protein components. ● Personalized Medicine & On-Site Production: Potential for point-of-care production of diagnostic proteins or patient-specific vaccine antigens. |
☞Cell-Free In Vitro Protein Expression Production Process
Our optimized process maximizes yield and functionality:
①Template Preparation: The gene of interest is cloned into a vector containing a T7, SP6, or other suitable promoter, or is amplified as a linear DNA fragment via PCR. mRNA templates can also be generated in vitro.
②Lysate Selection & Reaction Setup: Based on the protein's requirements (e.g., need for disulfide bonds, mammalian glycosylation signals), we select the optimal lysate:
● E. coli Lysate: High yield, cost-effective, ideal for cytoplasmic proteins, screening, and nnAA incorporation.
● Wheat Germ Lysate: Eukaryotic folding environment, suitable for producing complex, multi-domain eukaryotic proteins with higher likelihood of activity.
● Mammalian Lysate (e.g., HEK293): Capable of some post-translational modifications, best for proteins requiring mammalian folding pathways.
③Expression Reaction: The DNA/mRNA template is added to the master mix containing the chosen lysate, energy system, amino acids, and any supplements (DTT for reducing environment, chaperones, detergents). The reaction is incubated at the optimal temperature (typically 25-32°C) for 1-6 hours.
④Reaction Analysis & Processing: A small aliquot is analyzed by SDS-PAGE and/or Western Blot to confirm expression and estimate yield. The bulk reaction is then processed.
⑤Protein Capture & Purification: For tagged proteins, the reaction mixture is applied directly to an affinity resin (Ni-NTA for His-tag, glutathione resin for GST-tag). The simplified background allows for efficient one-step purification. For untagged proteins or specialized applications, the crude extract may be used directly or purified via alternative chromatographic methods.
☞Nebulabio Service Process
Our streamlined service ensures a fast turnaround from sequence to protein.
Process Steps Description:
● Project Consultation & Template Design: We discuss your protein, its intended use, and any special requirements (tags, nnAAs). You provide the gene sequence or purified DNA/RNA template.
● Lysate Selection & Reaction Setup: Our experts select the optimal expression lysate and configure the reaction conditions to maximize soluble yield for your specific target.
● Parallel Expression Reactions: The expression is carried out, often in parallel with small condition variations to optimize results.
● Expression Verification: We provide data confirming successful protein synthesis, including gel images and an initial yield estimate.
● Protein Purification: The target protein is purified from the reaction mixture using affinity chromatography or other appropriate methods.
● Basic QC & Delivery: The eluted protein is delivered in buffer, along with a report detailing the methods, yield, and purity assessment (e.g., SDS-PAGE analysis).
☞Accelerate Your Fc Fusion Biologic Development
Our Cell-Free Protein Expression Service delivers the agility your project needs, turning genetic information into functional protein in record time. Overcome the limitations of cellular systems and access a faster, more flexible path to protein for screening, engineering, and analysis.
Contact Nebulabio to discuss your project and learn how our cell-free platform can benefit your research timeline.
For more information, please contact us at info@nebulabio.cn or +86-15801534258.
