FAQ | CellFree Sciences

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What is the wheat germ cell-free protein expression system?

Cellfree Sciences offers a unique cell-free protein expression system based on extracts prepared from wheat germ. Special procedures have been developed to prepare high performance extracts for in vitro translation reactions. These allow rapid protein synthesis on different reaction scales and conditions under standard laboratory settings. We have built an entire portfolio based on this technology to serve different protein needs.


Why working cell-free to prepare proteins?

Working outside of the cell allows us to focus only on protein synthesis thus avoiding interference from other cellular processes. We are using this ability to develop dedicated reagent kits serving different protein needs. Furthermore, also proteins toxic in cells can often be expressed in cell-free systems.


What proteins can be made in a cell-free expression system?

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 in the kingdom of life. This includes large protein sets prepared from genomic resources holding human, mouse, Arabidopsis, and malaria cDNAs.


What is the size of proteins that can be expressed in the wheat germ system?

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.


What do I need to setup a cell-free protein expression system?

Cell-free protein expression experiments are easy to setup and do not require any dedicated laboratory equipment. Our kit manuals offer detailed information on how to perform protein expression experiments. Each kit provides all necessary reagents to prepare RNA from DNA templates and later to use the RNA for protein synthesis. We are also providing dedicated expression pEU vector set including a positive control vector for use with our expression system. A general introduction on the use of the wheat germ system was published in the literature: PMID: 33842544 (open access).


What is the template for a cell-free protein expression reaction?

Cell-free protein expression reactions require a DNA template, where our wheat germ system uses the SP6 RNA polymerase promoter to drive RNA synthesis. The template must further provide an enhancer to drive cap-independent translation reactions, where we are using the artificial E01 element. For optimal results, we advise to use our expression vectors of the pEU series to prepare expression templates for use in our expression system. We can also provide a protocol to prepare the expression template by PCR. However, working with a linear DNA template in protein expression reactions commonly gives lower protein yields as compared to using an expression vector.


Is codon optimization required for protein expression in the wheat germ system?

Most proteins have been successfully expressed in the wheat germ system when using large cDNA collections in template preparation. Thus, codon optimization is not necessary for working with the wheat germ system. However, when preparing new templates by gene synthesis, we recommend optimizing the sequence using the optimization options for wheat offered by most gene synthesis providers. These routines not only consider codon use but also optimize parameters like RNA stability and folding.


Which affinity tags can be used in the wheat germ cell-free system?

We are providing dedicated expression vectors for working with the Flag- (DYKDDDDK), GST- and His-tag. All of them have been successfully used when working with the wheat germ system. We are further offering special versions of our wheat germ extracts that were pretreated on a glutathione or a Ni resin to remove proteins from the extracts that could bind to such resins. These special G and H versions offer better protein purity when working with the GST- or His-tag. The use of other affinity tags in combination with the wheat germ system was described in the literature.


Does the wheat germ system promote post-translational modifications?

The endoplasmic reticulum was removed during extract preparation. Hence, there will be no protein processing and glycosylation of proteins expressed in the wheat germ system. Other post-translational modifications like phosphorylation, acetylation, or myristoylation have been observed in some cases, though we have no information on whether such modifications would match with native patterns.


Does N-terminal methionine processing occur in the wheat germ system?

Yes, wheat germ extracts contain a methionine aminopeptidase, the enzyme that removes N-terminal amino acids from proteins with a preference for methionine. N-terminal methionine processing is depended on the protein sequence and thus results may vary for different proteins.


Can signal peptides be used in the wheat germ system?

We advise to better remove signal peptides when designing expression templates. Since the endoplasmic reticulum was removed during extract preparation, there will be no protein processing and accordingly signal peptides will not be removed during expression reactions in the wheat germ system.


Can proteins made in the wheat germ system be dephosphorylated?

Proteins made in the wheat germ system can be dephosphorylated e.g., to study protein phosphorylation by mass spectrometry. Reference standards made in the wheat germ system have been dephosphorylated by incubation with lambda protein phosphatase for 30 min at 30 °C (Reference PMID: 33188775 open access).


Can the wheat germ system be used for protein labeling experiments?

We are offering our wheat germ cell-free system in an amino acid-free version (WEPRO8240 series). Using products of the WEPRO8240 series allows customers to use their own amino acid preparations in the translation reactions. We further offer dedicated products for the incorporation of stable isotope-labeled amino acids working together with our partner Cambridge Isotope Laboratories. These products are intended for preparing NMR samples or reference standards for applications in proteomics. NOTE, nonstandard amino acids commonly cannot be directly used in our expression system, because they are not recognized by any of the natural aminoacyl-tRNA synthetases.


Do you offer reagents for radioisotope labeling of proteins?

No, we are not offering dedicated reagents to prepare radioisotope-labeled proteins in in vitro translation reactions. However, tracer amounts of labeled amino acids can be added to regular expression experiments in the wheat germ system without changing the reaction conditions e.g., to quantify protein synthesis. For high incorporation rates, customers can purchase our amino acid free WEPRO8240 kits. These allow customers to prepare their own amino acid mixtures for use in the protein synthesis reactions including the option to use radioisotope-labeled amino acids.


Do you offer reagents for non-radioactive protein labeling?

We do not offer such a product at this point, but different reagents are available for non-radioactive labeling of proteins using the wheat germ system. Promega offers the "FluoroTect™ GreenLys in vitro Translation Labeling System" (Product Number: L500A) that uses a modified charged tRNA to randomly introduce a labeled lysine during protein synthesis. The FluoroTect™ GreenLys is a very powerful tool and can be used for every template having the codon for the charged tRNA. We can provide a protocol for quickly testing protein expression in our expression system using the FluoroTect™ GreenLys system. We added some FluoroTect™ GreenLys to our Premium ONE Expression Kit (Product Number: CFS-EDX-ONE) for testing.


Can the wheat germ system be used to prepare biotinylated proteins?

We have a method that uses the BirA biotin ligase to prepare mono-biotinylated proteins directly during cell-free protein synthesis. The approach requires that proteins are expressed with an added recognition sequence for the BirA ligase. Afterwards, the BirA ligase and D-biotin are added to the translation reaction to conduct the biotinylation reaction during protein synthesis. May be up to 90% of the synthesized proteins can be biotinylated in this process, however biotinylation rates may be changed by using different biotin concentration in the reaction. Mono-biotinylated proteins made by this process have been successfully used in several applications and could have principal advantages as compared to other approaches using randomly biotinylated proteins where biotin moieties could change protein surfaces and possibly protein folding.


CellFree Sciences is offering services to prepare mono-biotinylated proteins following the aforementioned process. Contact us for more information.


Are there biotinylated proteins in wheat germ extracts?

While it is sometimes stated that wheat germ extracts can contain biotinylated proteins, we have not observed this when testing our extracts in Western Blot experiments using a Horseradish Peroxidase-labeled Streptavidin for detecting biotinylated proteins.


What can I do when protein synthesis fails?

We are providing full technical support on the use of our expression system to assure customer success and have troubleshooting guides. Furthermore, our manuals provide advice on how to address problems when working with our expression system. Contact us for more help and support if uncertain about your results.


Do wheat germ extracts contain lipids?

We assume that some lipids remain in the wheat germ extracts at the end of the process. However, we do not have characterized or quantified remaining lipids for our different extracts.


Can lipids be added to translation reactions?

Yes, lipids can be added to translation reactions in the form of liposomes. CFS is offering special liposomes prepared from asolectin from soybeans for the preparation of membrane proteins. During translation reactions membrane proteins can enter the liposomes to form proteoliposomes.


What is the size of liposomes offered by CFS?

The size of liposomes may vary but the liposome preparations we are offering should have a distribution peak of about 150 nm based on scattered light intensity and should be monodisperse.


Can detergents be added to cell-free protein expression reactions?

The use of several detergents was described in the literature for working with wheat germ cell-free expression system e.g., to increase protein solubility. CFS can provide information on detergent use in our wheat germ system. Note, that the working concentration for each detergent must be experimentally determined as detergents can inhibit translation reactions when used at high concentrations. Note further, detergents can have effects on your protein of interest and may be difficult to remove.


Can cofactors be added to cell-free protein expression reactions?

It is the advantage of cell-free protein synthesis that cofactors can be added to the translation reactions to meet with protein needs. However, all additives should be tested at different concentrations for their impact on the translation reactions and the protein of interest. CFS can provide information on the use of metal ions in translation reactions.


Can Dimethyl Sulfoxide (DMSO) be added to cell-free protein expression reactions?

DMSO is a water-soluble organic solvent, that can be added to translation reaction up to a concentration of 1% (v/v). Used at a higher concentration will inhibit the translation reaction and lower protein yields.


What is the correct amino acid concentration in the translation reaction?

Our translation buffers contain each amino acid at a final concentration of 0.3 mM.


Is Dithiothreitol (DTT) used during translation reactions?

Our regular translation buffers contain DTT at a final concentration of 4 mM. Translation reactions must be performed under reducing conditions and hence DTT is required to maintain the reactions. Note, we are offering special reagents for preparing proteins having disulfide bonds, as disulfide bonds may not be stable at high DTT concentrations.


Can Ethylenediaminetetraacetic acid (EDTA) or Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) be used in translation reactions?

EDTA and EGTA are chelating agents that can bind metal ions. Since the Mg2+ ion concentration is critical for the translation reaction, they should not be added to the translation reaction.


What is the potassium concentration used during translation reactions?

Our translation buffers contain potassium acetate at the final concentration of 100 mM.


Do you have information on the endotoxin levels in your wheat germ extracts?

We have analyzed our standard WEPRO7240 extract for endotoxin contaminations and found 150-300 endotoxin units/ml. For purified proteins made with the WEPRO7240 wheat germ extract we found on average 0.1-0.5 endotoxin units/ml. Depending on the regulatory body, this would be an acceptable level for the endotoxin contamination when applied to medical devices.


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