Cell-Free In Vitro Protein Expression

As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for the expression of proteins including large proteins and membrane proteins, which are challenging for traditional expression systems. Creative BioMart has developed cell-free in vitro expression systems to meet the increasing demands for the protein synthesis of those particular protein types.

What is Cell-Free In Vitro Protein Expression?

Cell-free in vitro protein expression can be performed in so-called "coupled" systems running RNA transcription and protein translation at the same time in the same reaction mixture. It is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Only two basic conditions are required to establish the system: the template encoding the target protein and a reaction solution containing the necessary transcriptional and translational molecular machinery.

Cell Extracts Supply All Or Most of The Molecules of The Reaction Solution

RNA polymerases for mRNA transcription

Ribosomes for polypeptide translation

tRNA and amino acids

Enzymatic cofactors and an energy source

Cellular components essential for protein folding

Advantages of Cell-Free In Vitro Protein Expression

• Able to express proteins that are toxic to host cells
• High throughput, can express several different proteins simultaneously
• Reaction conditions are easy to change, allowing for protein modification control
• Ability to express different types of proteins
• Capable of producing cytotoxic proteins
• Simple operation and time saving

Service Content of Cell-Free In Vitro Protein Expression

1

Gene Synthesis Plus Codon Optimization

• Leading codon optimization system
• Analysis and optimization of rare codons, codon bias and other factors

Timeline: 1-2 weeks

2

Vector Construction

• Clone target gene into expression vector
• Plasmid sequencing
• Plasmid preparation

Timeline: 1 week

3

Protein Expression and Purification

• Expression condition optimization
• Protein expression evaluation
• Various protein purification methods

Timeline: 1-2 weeks

4

Optional Services

• Secondary purification
• Tag removal
• Endotoxin removal
• Activity assay
• Protein analysis
• Other more

Timeline: 1-2 weeks

5

Quality Control

• SDS-PAGE
• Western blot
• Mass spectrometry

Timeline: <1 week

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Creative BioMart offers a wide variety of cell-free in vitro systems. Selection of a cell-free system should be according to the biological nature of the protein, application and the template used for protein expression. Our team can guide you through the process of the cell-free expression if you would like to discuss more about your project.

Different Cell-Free Expression Systems

System	Advantages	Disadvantages
E.coli Extract	Established system Relatively low cost High translation speed Can be coupled with nanodisc assembly for membrane proteins Genetically modified strains available	No post-translational modifications of the eukaryotic systems Codon usage optimization is required
Wheat Germ	Established system Generally highest yield among eukaryotic cell-free systems High ratio of soluble expression Suitable for the synthesis of large proteins (∼200 kDa)	Relatively high cost due to time-consuming extract preparation Some background phosphorylation was observed in protein MS studies No glycosylation
Insect Cell Lysate	Suitable for the synthesis of large proteins Cap independent translation N-glycosylation possible Formation of disulfide bridges	Relatively new system Lower protein yield than E. coli or wheat germ systems Relatively high cost
Rabbit Reticulocytelysate	Established system Mammalian system Cap independent translation Often used in research	Sensitive to additives No glycosylation Co-expression of off-target proteins was observed Hemoglobin con. 90% of protein
Human Cell Lysate	Some protein modification	Low yield Sensitive to additives