Cell-Free In Vitro Membrane Protein Expression
Cell-free protein synthesis (CFPS) is a protein expression approach that enables production of a target protein without the use of living cells. The production of membrane proteins is a challenging task due to their hydrophobicity and their specific requirements in cellular expression systems. Unlike conventional cellular expression systems (e.g. bacterial, yeast or mammalian cells). So far, a considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters (SMTs) or eukaryotic G-protein coupled receptors (GPCRs) have been produced in cell-free systems in high amounts and in functionally active forms. Depending on the lysate system, cell-free protein expression can be categorized into: E. coli Cell-free Expression, Wheat germ Cell-free Expression and other Mammal Cell-free Expression System. In a simple biochemical reaction, CFPS generates desired proteins in just hours compared to traditional in vivo protein expression approaches that take several days or longer.
Principle of Cell-free Expression System
Cell-free in vitro protein expression can be carried out in so-called "coupled" systems, where both RNA transcription and protein translation are performed in the same reaction mixture. The cellular machinery required for protein synthesis is extracted from a cell-free system and placed in a test tube or reaction vessel as shown in the essential components of cell-free protein synthesis figure. These systems are comprised of three fundamental components:
- The genetic template (mRNA or DNA) encoding the target protein;
- A reaction solution containing the necessary transcriptional and translational molecular machinery;
- Energy sources (e.g., ATP, GTP) and cofactors (e.g., magnesium ions) are supplied to facilitate transcription and translation processes.
It is preferred for many applications in protein research, including protein labeling options and expression of difficult-to-express proteins such as membrane proteins and multiple protein complexes.
Figure 1. The essential components of cell-free protein synthesis. (Front Bioeng Biotechnol. 2019 Oct 11;7:248. doi: 10.3389/fbioe.2019.00248.)
![The essential components of cell-free protein synthesis. (Front Bioeng Biotechnol. 2019 Oct 11;7:248. doi: 10.3389/fbioe.2019.00248.)](http://www.betalifesci.com/cdn/shop/files/fbioe-07-00248-g001_{width}x.jpg?v=1721337485)
Cell Extracts for In Vitro Protein Translation Types of Cell-Free Extracts
The first CFPS experiments used cell extracts derived from E. coli. Thanks to its rapid growth kinetics, E. coli rapidly provides high protein yields, but some eukaryotic proteins were found to be insoluble after E. coli CFP. Progress in this field has led to the use of cell types such as wheat germ, Leishmania tarentolae, rabbit reticulocyte, insect cells, plant cells and human cells.
Each cell type offers distinct advantages. For example, wheat germ extract enables the translation of large proteins. Furthermore, wheat germ extract does not contain endogenous mRNAs, which could be translated at the same time as the target protein. Proteins expressed from wheat germ also retain their complex three-dimensional conformation, since they are derived from a eukaryotic source. The use of plant cell extracts increases yields up to 3mg/mL and enables complex proteins to be folded and glycosylated.
![Table 1: Comparison of four different cell-free expression systems](http://www.betalifesci.com/cdn/shop/files/unnamed_93cb41a1-4045-4126-9a1b-9246b5cbc2e0_{width}x.png?v=1721338109)
All four cell-free expression systems have their respective fields of application in which they perform best. E. coli, for example, produces large quantities of protein and is relatively tolerant of additives, but does not perform well with eukaryotic proteins. Wheat germ extracts or insect cell systems, such as Sf21, enable the translation of larger proteins while offering moderate yields. Finally, mammalian expression systems based on rabbit reticulocyte lysates allow the user to perform cap-independent translation while using a eukaryotic system. The disadvantages, however, are lower relative yields or increased sensitivity to additives.
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How Does Cell-Free Protein Synthesis Compare to Other Methods of Protein Production?
The Cell-Free Protein Expression System (CFPS) is significantly different from the traditional intracellular expression system (In vivo): the In vivo system requires the insertion of the target gene into a plasmid, which is then transformed into the host cell. After antibiotic screening, the transformed cells containing the target gene are grown in culture medium to produce the target protein. Finally, the target protein is extracted and purified by cell lysis and centrifugal separation. In contrast, the CFPS system directly accesses the protein synthesis components after cell lysis by first growing and lysing the cells, and the lysate is centrifuged to remove cellular debris and DNA. Then, essential components such as target gene templates, energy sources, cofactors, and salts are added to cell-free extracts, and protein synthesis proceeds directly. Finally, the target protein is obtained by centrifugation and purification steps.
The CFPS system eliminates the need for cell culture and screening steps and enables rapid protein expression, which is particularly suitable for proteins that are difficult to express in living cells or for high-throughput screening.The CFPS system is more efficient and flexible and, due to the absence of physiological constraints, dramatically improves the overall yield of functional, soluble, full-length proteins.
Figure 2. A comparison of a conventional in vivo system and a CFPS system. (Front Bioeng Biotechnol. 2019 Oct 11;7:248. doi: 10.3389/fbioe.2019.00248.)Cell-Free Protein Synthesis Applications
CFPS is well-suited for many applications. The speed and ease of CFPS facilitates high throughput protein expression in applications such as protein engineering, mutagenesis studies, and enzyme screening. In addition, CFPS is often used to generate proteins for biophysical and structure-function analyses. Other CFPS applications include the production of proteins that are toxic to host cells in vivo, expression of proteins with modified or unnatural amino acids, and incorporation of post-translational modifications (in some CFPS systems). Due to their robust performance, scalability and cost-effectiveness, CFPS systems have also been adopted in metabolic engineering and synthetic biology applications.
The utility of CFPS is likely to expand, due to the potential it offers for high-throughput, cost-effective production of large quantities of research-grade proteins.
Advantages of Cell-Free Expression of Proteins
Cell-free protein synthesis offers quite a few advantages. There listed a couple of benefits here:
- Cell-free reactions are easy to set up and take only several hours to express your protein. Optimization of reaction conditions can be done quickly and parallel by the use of small-scale reactions.
- Small handling volumes: mg amounts of soluble proteins obtainable from only a few ml of reaction volume.
- The open system makes it possible to add numerous components (natural or otherwise) to the reaction, e.g. reducing/oxidizing elements, chaperones, labeled amino acids or detergents. Direct manipulation of the chemical environment is possible. Proteins can be used in applications such as nuclear magnetic resonance (NMR); Reconstitution of membrane proteins in nanodisc systems in their nascent state.
- Eukaryotic cell-free lysates provide post-translational modifications such as core glycosylation or phosphorylation and natural membrane components (e.g. microsomes) for membrane protein insertion.
- Aspects such as toxicity, protease digestion or DNA matrix instability are not a problem, since the cells are no longer alive and the responsible cellular components have been eliminated.
Proteins and complexes with multimeric conformations can be expressed, and it is possible to affect the ratios of their respective subunits by titration of the DNA matrix.
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. Beta Lifescience has developed cell-free in vitro expression systems to meet the increasing demands for the protein synthesis of those particular protein types.
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Service Details for CFPS-based Membrane Protein Expression
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FAQs
Yes. A cell-free system is a set of enzymatic cascade reactions, and its open characteristics allow the addition of required elements to achieve eukaryotic protein expression. The functions of some target proteins such as antibody fragments (scFv and VHH) are not dependent upon post-translational modifications. If these proteins are expressed by cell-free and HEK293 systems, there is no significant difference in functional activity.
One drawback of eukaryotic CFPS systems include relatively lower protein yield compared to E. coli. Cell-free expression systems are often used in smaller reaction volumes, which may limit the production process. Additionally, the use of small volume extract preparation is not cost efficient.
The wheat germ cell-free protein expression system is well established and was used over the years to successfully express all types of proteins from different viruses, bacteria, parasites, plants, and animal species covering the entire kingdom of life. This includes large protein sets prepared from genomic resources holding human, mouse, Arabidopsis, and malaria cDNAs.
We have a track record of synthesizing proteins from 10 kDa to 360 kDa, where a protein of 360 kDa is an exceptional case. Proteins smaller than 10 kDa may be made as well, but we do not have much experience working with such small peptides in our expression system.
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