Cell-Free Protein Expression

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VectorBuilder provides high-quality protein production service through our Cell-Free Protein Synthesis (CFPS) platform. CFPS enables in vitro protein expression outside of living cells. The process of recombinant protein expression through the CFPS has been optimized and streamlined, allowing for completion in as little as five days. This significant acceleration enhances the efficiency of your workflow.

Highlights

Fast turnaround time
High level of control over the reaction conditions
Suitable for high-throughput production of a range of target molecules
Adaptable for producing antibody fragments that are difficult to express in cellular systems

Service Details

Cell-Free_Protein_Expression_Service

Our workflow starts from the design and construction of the expression vector, which will be used as the template in the CFPS. E. coli cells are lysed, and the crude extracts are collected from cultured cells. The lysate, which contains the necessary cellular machinery, will be further processed, and necessary components such as energy molecules, amino acids, and DNA or mRNA template will be supplemented to initiate transcription and translation for protein synthesis. The process of cell-free synthesis, protein purification, and QC can be finished in as fast as 1 day.

Shipping and storage

Most of our custom recombinant proteins are delivered as frozen liquid on dry ice. Upon receipt, they should be stored at -20°C to -80°C under sterile conditions. Recombinant proteins typically remain stable for up to a year if stored properly. Additionally, we recommend aliquoting the recombinant proteins upon receiving and avoiding freeze-thaw cycles.

Quality control (QC)

All vectors cloned by VectorBuilder come with a 100% sequence guarantee. All recombinant proteins produced by VectorBuilder undergo stringent quality control. Default QC for most systems includes 1) validation of the protein expression vector by restriction digestion analysis and Sanger sequencing; and 2) determination of protein concentration and purity by A260/280 measurement and SDS-PAGE. Common additional QC services are shown in the table below, which can be provided upon request.

Additional QC Services	Method
Endotoxin test	LAL
Protein characterization	Western Blot
	Intact MS (reduced)
	SEC-HPLC
	SEC-MALS
	Protein N-terminal sequencing
	Host cell protein test
Tag removal	Protease digestion
Kinetics and affinity analysis	Octet
Kinetics and affinity analysis	Biacore
Pathogen testing panel for rodents

Technical Information

Case study

High-throughput VHH synthesis by CFPS. scFv synthesis by CFPS. The purity was determined to be >90% by SDS-PAGE.

Figure 1. (A) High-throughput V_HH synthesis by CFPS. The purity was determined to be >90% by SDS-PAGE. (B) scFv synthesis by CFPS. The purity was determined to be >90% by SDS-PAGE.

How to Order

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Customer-supplied vectors

If customer-supplied materials are needed, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder, which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.

Resources

FAQ

Which recombinant protein expression system should I choose?

All recombinant protein expression systems have their pros and cons which should be taken into consideration while selecting the optimal system for your project. The table below summarizes the pros and cons of each system.

Recombinant Protein Expression System	Pros	Cons
Bacterial	Cost-effective Short production time Technically simple Can be scaled up easily High protein yield	Proteins lack post-translational modifications Codon usage is different from that of eukaryotes Difficult to express certain proteins due to the accumulation of inclusion bodies Some proteins are toxic and can inhibit bacterial growth
Mammalian cells	Produces proteins in their most natural state with all necessary post-translational modifications and proper folding Suitable for secretory and membrane proteins Most appropriate for producing therapeutic proteins	Long production time Requires complex growth conditions Scaling up can be difficult Not suitable for intracellular proteins due to low yield
Insect	Performs majority of complex post-translational modifications and protein folding Suitable for secretory, intracellular, and membrane proteins Can be used for producing large protein complexes High levels of protein expression compared to other eukaryotic expression systems Highly scalable due to high-density, suspension cell cultures	Long production time Requires complex growth conditions Technically challenging
Cell-free system	Time-efficient synthesis completed in 3 hours No need for live cells in the production of toxic, complex, or unstable proteins Suitable for high-throughput protein expression and screening Easy procedure compatible with automated processes Easy to optimize the conditions	Limited modifications are available

Which protein tag should I use?

Tags are frequently utilized for recombinant protein production. They streamline the purification process, and certain tags have demonstrated improvement in protein solubility, yield, or purity. If the tag is attached to the protease cleavage site, it can be removed after purification, and the efficiency of cleavage varies depending on the target protein. The careful selection of an appropriate tag is crucial for downstream protein expression and purification. The table below provides an overview of commonly used tags along with their advantages and limitations.

Tag	Commonly Applied System	Advantages	Limitations
GST	Bacteria, insect	Largely preserve the native structure of the protein Enables protein purification under mild conditions Easy cleavage Enhances the solubility and expression of the protein	Large tag size Dimerization may impact the target protein Not suitable for purifying proteins under denaturing conditions
His	All	Small tag size, therefore less impact on the protein Low cost for metal-affinity chromatography Low immunogenicity Suitable for purifying proteins under denaturing conditions	Other endogenous metal-binding proteins in microbial hosts may be co-purified, therefore optimization is usually required Does not facilitate protein folding and solubility
SUMO	Bacteria, insect	Facilitates protein folding Enhances the solubility and expression of the protein	May undergo non-specifically cleavage by other bacterial proteins Not suitable for purifying proteins under denaturing conditions
Flag	Mammalian cells, insect	Small tag size, therefore less impact on the protein Easy detection Low non-specific binding	Poor efficacy for purification
MBP	Bacteria, insect	Enhances the solubility and expression of the protein	Large tag size Not suitable for purifying proteins under denaturing conditions
Fc	Mammalian cells, insect	Enhances the solubility and expression of proteins Suitable for secreted protein	Large tag size

For more information regarding tags, please visit protein tags.

Can I synthesize eukaryotic proteins using the cell-free synthesis platform?

Yes, CFPS involves mixing a cell extract containing the necessary machinery for protein synthesis with other essential components. By incorporating components derived from eukaryotes, the synthesis machinery closely resembles that in eukaryotic cells, enabling the accurate synthesis of eukaryotic proteins.

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