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Does ProteoExpert provide information how the amount of soluble or functional protein product can be increased?

ProteoExpert only addresses questions of protein expression yield, not solubility or function. However, the program will scan your protein sequence for regions that are likely to be involved in interactions with GroE chaperones and could therefore be important for proper folding. If ProteoExpert informs you that such a domain was identified, solubility and functionality of your expressed protein may be increased by adding RTS GroE Supplement to your expression reactions.

Are the primers and conditions ProteoExpert suggests checked for performance in PCR?

Primer sequences and PCR-conditions suggested by ProteoExpert should be considered as general recommendations and may have to be adapted to your specific experimental conditions.

What do I need if I want to use ProteoExpert?

Similar to other web-based services (e.g. online banking), the use of ProteoExpert requires special access numbers. From May 2007 on this service is only available directly through Biomax.com.
One number is needed per sequence submitted to the ProteoExpert server.
The sequence you want to submit must be available in text or FASTA format without start or stop codons. For experimental procedures downstream of ProteoExpert calculations (generation and small-scale expression of sequence variants), we strongly recommend to use RTS E. coli Linear Template Generation Sets and RTS 100 E. coli HY Kits www.proteoexpert.com and www.proteinexpression.com for details).

When I do the RTS reaction, a large fraction of the expressed protein is in the pellet. Do you have a protocol to get the protein into the supernatant?

There are several ways to optimize solubility:
- use chaperones, e.g. the RTS GroE Supplement , for the correct folding of proteins between 20 and 60 kDa;
- add  detergents for hydrophobic proteins. Unfortunately there is no way to predict which detergent will be successful;
- add cofactors;
- try different tags, especially  MBP fusion, and tag positions. MBP-fusion proteins are often very soluble. The fusion vector contains a Factor Xa cleavage site that allows you to later purify your protein away from the MBP (Maltose Binding Protein).
- vary reaction time;
- vary reaction temperature (14-37°C). The higher your reaction temperature, the more likely it is to obtain aggregated proteins.

Can the RTS pIVEX vectors be used for protein expression in E. coli systems?

The pIVEX vectors are designed for high-level expression of proteins in the cell-free RTS E. coli system. They have not been tested nor optimized for expression in in vivo systems.
We therefore can not recommend the use of the RTS pIVEX vectors with expression systems other than RTS.
In principle they should work in E. coli. However, the pIVEX vectors are not IPTG inducible and have a high copy origin. This may result in constitutive protein expression which might harm the host cell.

The GFP from the GFP control reaction is partly precipitated. How can I quantify expression yield?

The GFP control reaction is only intended to be a qualitative control to see whether the expression worked or not. It is dangerous to quantify GFP via fluorescence because of the oxidative maturation that is needed. Quantification will depend very much on the time that has passed between expression and quantification as well as on storage conditions (oxygen supply etc.). If quantification is desired, because of the above-mentioned restrictions, it is strongly recommended to quantify total GFP (if the customer really wants to do it) on a Western blot using anti-his6 antibodies with the rGFP (Cat.No. 1814524) as a reference instead of using fluorescence.

Can linear templates be used in RTS?

Linear templates can be used in the RTS 100 E. coli HY Kit. For scale-up in the RTS 500 HY and the RTS 9000 HY Kits circular templates have to be used.

Is it possible to add DMSO to a RTS expression reaction?

It is possible to add DMSO to the RTS reaction. We have good experience with 3-5% DMSO. This corresponds to 1.5-2.5 µl 100% DMSO in a 50 µl reaction. This becomes relevant especially when GC-rich templates that have been generated by PCR shall be used, because often DMSO is added to the PCR of GC-rich DNA. Thus, the PCR does not need to be purified but can be used directly in RTS 100 E. coli HY despite the addition of DMSO.

A white precipitate is observed after addition of SDS-PAGE sample buffer to the RTS expression reaction.

The problem arises most probably due to precipitation of SDS with the potassium salts from the RTS reaction. Please compare the note on page 3 in our RTS Application Note Protein Analysis and Purificaton. The solution to the problem would be to either dilute the reaction mix 4-fold prior to sample preparation or to precipitate the sample with acetone as described in the Tips and Tricks section on our Special Interest Site.

Does the amount of template DNA affect the reaction yield?

The amount of template DNA has not got a significant influence on the yield in RTS 100 E. coli HY in the concentration range we recommend. This is, however, only true for pure DNA preparations. High amounts of low quality DNA may inhibit the reaction. Too much pure DNA can also inhibit the reaction. Absolute values of minimum/maximum concentrations can not be given, this depends on the DNA quality and on the size of the target gene.

Why can the use of MBP-fusion protein increase the solubility of the target protein?

The solubility of a protein is a matter of sequence context. MBP does ameliorate the solubility. This was found out empirically. There are citations speculating about MBP itself functioning as chaperone but these are only hints.

Does the MBP-fusion protein affect the activity of the enzyme to be expressed?

MBP may affect the activity of the expressed protein. This depends on the expressed protein and can not be predicted. The MBP-fusion protein can be removed because of a Factor Xa restriction protease cleavage site in the pIVEX MBP Fusion Vector and the RTS E. coli Linear Template Generation Set, MBP Fusion.

Is there any wild type or mutant beta-Galactosidase present in the RTS E. coli lysate?

The E. coli strain used for lysate production has a wild type beta-Galactosidase gene, therefore the protein will most probably be present in the lysate but has not been determined.

Is EDTA present in the RTS E. coli lysate?

EDTA is not present in the RTS E. coli lysate. Furthermore, it can not be added to the reaction because it inhibits the reaction.

Do you have recommendations for the conversion of linear templates from the RTS E. coli Linear Template Generation Sets to circular templates for expression in RTS 500 and RTS 9000?

Cloning procedures to convert linear to circular templates are described in the RTS Application Note  "From linear to circular expression templates".

Is it necessary to purify the protein produced in RTS?

The necessity to purify the product strongly depends on the type of assay the customer wants to perform.
The product contains not only the protein of interest but also E. coli proteins necessary for transcription and translation of the template that are present in the lysate. Nevertheless, the product will be of much higher purity than from an in vivo expression. The background protein content is approximately 10 mg/ml.

Do you have recommendations for the expression of membrane proteins in RTS?

Feedback regarding expression of membrane proteins in RTS is still very limited. This is a summary of what we know so far or what is supported by the state-of-the-art of membrane protein biochemistry. We think there is no principle limitation regarding expression of membrane proteins in RTS. We have feedback from German customers that even complex membrane proteins (= many membrane domains) can be expressed. This means, that a substantial amount of protein can be synthesized, especially when using ProteoExpert for yield optimization. Membrane proteins tend to aggregate immediately and can not be expected to be soluble under standard conditions. Be aware, that the class of membrane proteins is extremely heterogeneous. There won't be standard protocols. But one could think of screens, identifying, which approach could be applied best, for a given protein. The more you know about the target protein the better the probability to find the right experimental track. Different factors should be considered, when looking for technical solutions:

Number of transmembrane domains:
Membrane proteins with few (1-2) transmembrane domains (e.g. receptor kinase type) could potentially be solubilized by addition of detergent. Often one can get some hints from literature, describing solubilization of similar proteins. E.g., if a similar receptor has been solubilized with Triton-like detergent, this could potentially be tried. Proteins with many transmembrane domains (e.g. GPCR's, transporter or ion channels) are known often to be dependant on phospholipids in order to exhibit correct conformation and function. So far we have no own experiences in this field. Generally there are two ways to try: a) stabilization during synthesis (detergent, chaperones; a nice effect we could observe in some cases is, that detergents or chaperones somehow have a positive influence on synthesis rate) or b) renaturation after synthesis, by solubilization/reconstitution techniques, as described in the literature. However, this normally requires quite a bit expertise in this field. We can refer you to our RTS Application Note "Detergents in RTS".

Subunit structure:
Often membrane proteins are composed of several or many subunits, interacting with each other and stabilizing each other. This could be an explanation, why individually expressed subunits or domains may be less soluble than expected. The use of a suitable detergent (could be different types for different proteins) may stabilize exposed hydrophobic surface areas. Replacement of hydrophobic amino acids in these areas by more hydrophilic ones could also have a positive effect. It is clear that this is not an approach for proteins with unknown structure, but for quite a number of proteins information from related proteins can be used.

Domain structure:
For functional or structural evaluation of membrane proteins it could be faster to re-design a protein, either by eliminating the transmembrane domain (if there is only one or few) or by individually expressing extra-cellular or intra-cellular domains. Using this approach, chaperons can play an important role. The Expression PCR approach can help to rapidly identify and optimize the most suitable domain boundaries. Dealing with extra-cellular domains, the factor of disulfide-bonding and glycosylation (=> solubility) has to be considered.

Modifications:
Glycosylation of extra-cellular domains can have an effect on solubility of a domain or a whole protein. Often putative glycosylation sites (serine-/threonine-, or asparagine residues) are mutated to more bulky, charged amino acids. A replacement of surface-located residues often does not strongly interfere with function.

What can I do to improve the purification/elution yield of MBP fusion proteins?

Elute with up to 500 mM Maltose.
Spin the columns.
Use the His6-tag for purification.
Mix the protein solution directly with the matrix (batch procedure)
Incubate an aliquot of the expression reaction at 30°C for 16-20 hours and check the protein content before and after the incubation. This shows whether the yield is decreased because of proteolytic degradation rather than adhesion to the purification matrix.

How can one maintain the solubility of the target protein in a GroE system, once the protein has entered the into the affinity matrix?

GroE does not function by keeping the protein in solution but rather folds proteins between 20 and 60 kD. Therefore, once the chaperone has fulfilled its task, it is not necessary to have it in the solution any more. It should therefore be no problem to remove the chaperone during the purification.

Have universal expression vectors been tested in RTS?

Universal vectors for the expression of proteins in different pro- and eukaryotic systems have not been tested in RTS.
Since the commonly used vectors are based on the T7 system for expression in E. coli they should in principle work in RTS. However, the regulatory sequences of the initiation region are not specifically optimal for E. coli in vitro expression and the expression yield with these vectors in RTS may therefore be rather low.

Why do we propose a one-step control reaction in the RTS E. coli Linear Template Generation Set, His6-tag?

In the RTS E. coli Linear Template Generation Set, His6-tag the T7 Promoter primer and T7 Terminator primer bind to the regulatory regions of the pIVEX vectors and therefore also in the GFP Control Vector. The primers can therefore be used directly to check whether the PCR in principle works. The expected fragment size is 978 bp. However, this PCR does not control whether the DNA (vials 3 and 4) provided in the Kit work.
The sequences have been changed slightly in the RTS E. coli Linear Template Generation Sets, HA-tag, AviTag and MBP Fusion to avoid by-products originating from pIVEX as a template in the first PCR that may be transferred to the second PCR. The T7 Promoter primer and T7 Terminator primer do not bind to the regulatory regions of the pIVEX vectors any more. Therefore, the control reaction here is a two-step control reaction and primers for the first PCR have to be ordered separately.

Do you have a protocol for the biotinylation of AviTagged proteins after the RTS expression reaction?

It is possible to perform the biotinylation after the expression reaction. The conditions should be the same as for the co-translational biotinylation, i.e. RTS Expression reaction:
Biotinylation Mix = 10:1 (same as for direct biotinylation), incubation at 30°C for 2-4 hours (RTS 100 E. coli HY reaction). However, this procedure is not advisable because it prolongs the time until the protein is "ready" and some proteins may not be stable at 30°C in the reaction mix that long.

After TA cloning I see a lot of mutations in the overlap region generated with the RTS E. coli Linear Template Generation Sets.

Please use HPLC purified primers or try another synthesis company.

Is it possible to add a suspension of phospholipid vesicles in the RTS expression reaction in the hope that the expressed membrane protein inserts into the vesicle bilayers?

In principle, phospholipids should work in RTS. However, so far, we have not looked into the use of phospholipids. Use the RTS 100 E. coli HY Kit and run expressions with the template as well as the control vector using different concentrations of phospholipids. It is very important to have a sensitive assay for the target protein in place (e.g. good working Western Blot or activity assay). Do not use heavily positively charged phospholipids like they are used in transfection, since they bind to the DNA and may prevent expression. Non-active incorporation of membrane proteins into membranes can usually only be expected of proteins which - are membrane-associated - have just one TM (transmembrane domain). Otherwise the proteins follow a pathway in order to be incorporated into the particular membrane which includes e.g. a stretch of amino acids at the N-terminus of the particular protein and cell-internal factors like SRP (signal recognition particle) and others. However, there is quite a gap of knowledge how proteins get spontaneously incorporated into membranes, especially under translation conditions.

The expressed protein precipitates as soon as it is mixed with the buffer used for purification via Ni-NTA columns.

Check the pI of the protein. If it is very close to pH 8.0 try to use a buffer with a different pH. Like this, the protein will be charged and less likely to aggregate. The lower pH limit that still allows binding of the protein to the Ni-NTA column appears to be approx. pH 6.3. Proteins start to elute from the column at pH 5.9, although this does of course also depend on the individual protein.

Add different substances to the solution/buffer to increase solubility of the protein:
2 M NaCl, 50 mM CHAPS, 50% glycerol, 8 M urea, 6 M guanidine hydrochloride, 0.1-2% Triton X-100 (although this has a high absorbance at 280 nm, therefore disturbs protein detection and cannot be removed by buffer exchange procedures), 0.1-2% Tween 20, 20 mM 2-mercaptoethanol, stabilizing cofactors specific for the protein.

Try purification at higher or lower temperature.

Keep in mind that aggregation of the protein can indicate that it is not folded properly and hydrophobic regions that should be hidden inside of the protein are exposed externally and let the protein molecules form aggregates. Therefore, the protein may not be functional. It is, however, possible that the protein folds correctly during the purification procedure or once it is in its optimal assay buffer, especially if the "overall", loose conformation was alright but needed to be fixed.

Why is the ATG of the gene not needed in the sequence of the forward primer for the RTS E. coli Linear Template Generation Set, MBP fusion?

The ATG is not needed in the forward primer because the RTS E. coli Linear Template Generation Set, MBP fusion adds a N-terminal fusion to the gene of interest. This means that the ATG and all other sequence elements necessary for transcription and translation are included in the sequences that are added to the gene of interest via the overlap extension PCR. The ATG provided by the original sequence is therefore not necessary for a successful transcription/translation reaction. Furthermore, problems like internal initiation can be avoided if the ATG from the gene of interest is not included in the expression template. Our researchers found that it is safer to eliminate this codon. However, if you want to keep it the reaction will most probably also work.

Is the LacI repressor present in the RTS E. coli lysate?

The LacI repressor is not present in the RTS E. coli lysate. When the LacI repressor is present on the plasmid used as a template it is advisable to add 0.1-1 mM IPTG. Higher concentrations of IPTG may be inhibitory.

The Tm for primers calculated by ProteoExpert is much lower than that calculated by the producer of the primers. What is the cause of this lower Tm generated by ProteoExpert?

The primers that are suggested by ProteoExpert contain a gene specific part (including the silent mutations) and a part that creates the overlaps that are needed in the second PCR with the RTS E. coli Linear Template Generation Sets. The melting temperature is calculated for the gene-specific part which is the part that has to anneal with the template in the first PCR. The Tm for the whole oligonucleotide is of course higher.

Is it possible to add SDS to RTS expression reactions?

The use of SDS is not recommended in RTS because it strongly disturbs the reaction.

How long can the GFP produced in RTS expression reactions be stored and what are the storage conditions?

GFP is stable for at least a year in the reaction mix at -20°C. At 4°C, however, the activity decreases and is completely lost after 1-2 weeks.

What is the size of the GFP control protein?

27 kDa.
The protein contains a C-terminal His6-tag for detection on Western blots.

Do we have a list of traditionally "hard to produce proteins" that have been successfully expressed in RTS?

There are a couple of proteins included in our expressed proteins list which are lethal in E.coli:
-  Binding protein for Growth Factor
-  Microtubuli binding protein (MID 1) RTS 500 - Maximize the Possibilities in Protein Expression
-  p120 polymerase.

Others are insoluble or produced as inclusion bodies in E.coli:
-  Interleukin-2 Cell-free Expression of Human Interleukin 2 (IL-2) with the RTS 500 E. coli Circular Template Kit
-  MIA Recombinant Expression of the Melanoma Inhibitory Activity (MIA) Protein Using Various Rapid Translation System  Modifications

Which tags are available in the RTS product portfolio?

We offer the following tags and fusion proteins for use in RTS:

His6-tag (pIVEX vector and RTS E. coli Linear Template Generation Set) for easy detection and purification.
HA-tag (pIVEX vector and RTS E. coli Linear Template Generation Set) for easy and sensitive detection and purification.
GST Fusion protein (pIVEX vector) for easy purification, immobilization or protein-protein-interaction studies.
MBP Fusion protein (pIVEX vector and RTS E. coli Linear Template Generation Set) for enhanced solubility.
AviTag (pIVEX vector and RTS E. coli Linear Template Generation Set) for easy monobiotinylation and use in combination with the streptavidin system for highly sensitive applications such as protein-protein interactions, detection and purification.

Which technologies are used in RTS?

For a comprehensive overview of the technologies used in RTS please have a look at the RTS Technologies section on our RTS Special Interest Site.

Where can I find pIVEX vector maps and RTS E. coli Linear Template Generation Set sequences?

Please refer to the following links for information about RTS E. coli Linear Template Generation Sets and pIVEX vectors and the respective sequences.

Where can I find a list of literature dealing with RTS?

Please have a look at the Literature and Resources section on our Special Interest Site. This section contains research articles, Biochemica articles, Application Notes, results, an expressed proteins list, Tips and Tricks, pack inserts, vector maps and sequences. Furthermore, abook containing many articles dealing with cell-free translation is available: Alexander S. Spirin (Ed.): Cell-Free Translation Systems.

Is there a newsletter with regularly updated information about RTS?

Please register for the RTS eNewsletter. You will receive an email every few months keeping you up-to-date about the RTS products family.

What is the minimum size of proteins that can be expressed in RTS?

When RTS 500 or RTS 9000 is used for the expression of proteins much smaller than 10 kDa it is possible that the protein will diffuse into the feeding compartment and will have to be purified from both the reaction and feeding compartments of the device. For expression of proteins smaller than 10 kDa and peptides, we therefore suggest to add a fusion partner such as GST or MBP to the protein/peptide of interest. Another strategy for expression of peptides is to express the peptide as a concatamer. This may also give a higher yield of expressed protein as opposed to the expression of one peptide. The smallest protein we tested was a peptide with 5 kDa (approx. 40 aa) which has been synthesized with a rather good yield.

Which metal ions can be added to RTS reactions?

Addition of copper up to 1 mM is possible.
Addition of nickel up to 0.1 mM is possible.
Addition of zink up to 1 mM is possible.
Calcium can be added in soluble form (e.g. calciumchlorid-hydrate). Concentrations in the µM range should be possible. The maximal concentration was not evaluated.

Which concentration of chaperones shall be added in RTS reactions for initial experiments?

Add chaperones in a concentration of 1-10 µM. The best concentration within this range has to be tested for every system. We recommend to start with trying 10 µM.

Regarding the maximum concentration of chaperones that can be added: Chaperones can be added at physiologic concentrations or a bit higher - the respective concentrations for the individual chaperones should be retrieved from the literature, depending on which chaperone shall be used.

Expression of genes with a high GC percentage can be problematic in E. coli. Is it possible to express such genes in RTS?

It is possible to express proteins with a high G/C content with RTS. We have successful expression examples (e.g. viral and human genes).
Nevertheless, sequences with a higher G/C content cause more problems. Most probably expression problems are due to mRNA secondary structures. Although generally spoken there is a good chance to get expression with an N-terminal His6-Tag, we suggest to try different tags and tag positions to increase the chance to get a good-expressing construct. Another possibility is to use ProteoExpert for optimization of the sequence for expression yield.

Is it possible to do a time course of the synthesis process in RTS 500 E. coli HY and remove aliquots at different time points without affecting the reaction?

It is possible to take aliquots at any time during the synthesis. Furthermore, this is one of the big advantages of the RTS 500 format! For example, take 6-8 times an aliquot of 20 µl. This is enough for a control on SDS-PAGE and Western blot.

Is one labeled amino acid (35S-methionine, start codon) enough for detection of a 36 amino acid peptide?

The total number of labeled residues in the protein is not as critical as the total yield of your protein. Therefore, if the expression yield is high, one labeled residue is enough for detection whereas with a low expression yield you need more than one labeled residue to see you protein.

Can the problem of inclusion body formation in E. coli be overcome using RTS? Is the formation of product aggregates during "in vitro" reaction possible, even if this wasn't the case in E. coli itself?

RTS uses an E. coli extract as the in vitro translation system. Therefore, in general, proteins that are expressed in this system have the same tendency to be soluble and functional, or insoluble and non-functional, as in E. coli. There are, however, several examples of eukaryotic proteins which form inclusion bodies in E. coli (e.g., human interleukin-2), that are expressed as soluble active proteins with RTS using standard conditions. Another great advantage of the ""open"" RTS system is that the transcription/translation reaction can be continuously monitored, and components that enhance solubility such as detergents, cofactors and/or proteins that assist the correct folding of proteins (molecular chaperones), can be added to the system or expression can be terminated before precipitation occurs. For optimization, we recommend the RTS 100 E. coli HY kit and the RTS GroE Supplement.

Do you have a Troubleshooting Guide for RTS?

Yes. RTS Trouble Shooting Guide.

When is agarose gel purification of a PCR product possible, when is it inhibitory for the expression in RTS?

Agarose gel purification prior to protein expression should be avoided. This means:
If you prepare your expression template using 1-step PCR: avoid gel purification.
In case of 2-step PCR: gel purification after the first PCR is possible, but agarose gel purification after the second PCR inhibits in vitro protein synthesis.

After cloning of linear templates generated with RTS E. coli Linear Template Generation Sets two T7 promoters may be present on the plasmid. Is this going to interfere with the expression yield?

There is no negative influence on expression yield if the two promoters are in the same orientation. However, if they are in opposite direction, antisense mRNA may be generated and diminish the expression yield. Therefore, we highly recommend to sequence the cloned PCR product and check the orientation in the vector.
More details can be found in the RTS Application Note " From linear to circular expression templates".

Do you have protocols for the sample preparation for SDS-PAGE after the expression reaction?

The RTS Application Note Protein Analysis and Purificaton contains a protocol for sample preparation. As the Reaction Solution of the RTS HY kits contains polymers that interfere with the separation of proteins with an apparent molecular weight between 20 kD and 30 kD we suggest to precipitate the samples with acetone prior to the addition of SDS-PAGE sample buffer. This is described in the  Tips and Tricks section on our Special Interest Site.

Is it possible to perform glycosylation and other post-translational modifications in RTS?

RTS E. coli lysate does not contain the membrane compartments and the enzymes that perform post-translational modifications in eukaryotic cells.
Glycosylation is not possible even if eukaryotic microsomes (e.g., dog pancreatic) were added, because the prokaryotic homologue of the signal-recognition particle (SRP) in the E. coli lysate is not able to dock onto the eukaryotic SRP receptor protein. However, because RTS is an "open" format, chemicals and enzymes that could potentially perform post-translational modifications may be tested directly in the expression reaction or following protein purification.

The His6-tagged protein does not bind to the Ni-NTA column.

Possible cause	Recommendation
The His6-tag is not present.	a) Check the sequence and reading frame. b) Check for possible internal translation starts (N-terminal tags) or premature termination sites (C-terminal tags).
The binding conditions are incorrect.	a) Make sure that the correct buffers and pH have been used. b) Decrease the concentration of imidazole in the binding buffer. c) Ensure that there are no chelating or reducing agents present. d) If reusing a column, make sure it has been regenerated correctly.
The tag may be inaccessible under native conditions.	a) Put tag on another part of the protein (N- or C-terminus, respectively). b) Purify protein under denaturing conditions (e.g., sodium phosphate buffer, pH 8.0 with 8 M urea or 6 M guanidinium hydrochloride; optionally, add 10-500 mM imidazole to increase binding specificity).
The tag may be degraded.	Include protease inhibitors and perform purification at 4°C
The cloumn capacity is exceeded.	Apply less fusion protein to the column.
The binding time is too short.	Use Ni-NTA Agarose in batch format to prolong the incubation time.

The fusion protein does not elute from the Ni-NTA column.

This may be due to one of the following causes:

 

 

Possible Cause	Recommendation
The column may not have been correctly charged with nickel ions.	Repeat the column preparation steps.
The elution conditions are too mild.	a) Increase concentration of imidazole in the elution buffer (>400 mM). b) Use gradient with increasing concentrations of imidazole. c) Carefully lower pH to create more stringent elution conditions. Note: Do not use a pH below pH 3.5 because low pHs will strip metal ions off the column. d)Try elution overnight at room temperature or at 4°C: Fill the column with elution buffer, incubate over night and wash out the eluted protein with elution buffer the following day.
The fusion protein may be precipitating.	a) Purify protein at room temperature or at 4°C: Fill the column with elution buffer, incubate overnight and elute the protein with elution buffer the following day. b) Add solubilizing reagents, e.g., 2 M NaCl, 50 mM CHAPS, 50% glycerol, 8 M urea, 6 M guanidine hydrochloride, 0.1-2% Tween 20, 0.1–2% Triton X-100. Note: Triton X-100 has a high absorbance at 280 nm, and cannot be removed by buffer exchange procedures. c) Add reducing agents such as 2-mercaptoethanol to help solubilization. d) Perform binding and elution in batch format to avoid high local concentrations of protein.

Is there any vector or method to create a factor Xa site at the C-terminal side of the MCS of pIVEX vectors?

This may be due to one of the following causes:

We do not offer any pIVEX vector with a C-terminal factor Xa cleavage site. However, it should be no problem to add this site to the gene of interest by PCR mutagenesis. The reverse primer for template generation would have to be designed as follows:
5'-vector - FXa-sequence - your sequence

Factor Xa cleaves after the sequence: Ile-Glu-Gly-Arg
In our vectors, this corresponds to the nt-sequence: ATC GAA GGC CGC

However, these four amino acids will remain at the C-terminus of the protein of interest. Therefore, consider whether it is really necessary to remove the tag, because it is not very big anyway and you then will not have to worry about removal of the protease from the protein preparation.

Is there a maximum length of the insert that can be cloned in pIVEX vectors?

We have not tested the size limit of the pIVEX vectors. It should be similar as for all plasmid vectors, i.e. might become difficult to transform when the insert exceeds 5-6 kb. The largest protein tested so far was beta-galactosidase with a molecular weight of 116 kDa, corresponding to an approx. gene size of 3 kb. No problems with cloning have been observed in this case. Please compare RTS Application Note 2.
If you want to express large proteins you should increase the amount of template to maintain a high copy number of the gene in the reaction.
In case you plan to clone and express an operon please consider to clone the individual genes separately and perform coexpression using more than one plasmid in the reaction. This leads to more flexibility to adjust the amounts of the individual proteins via the template concentration because expression from an operon does not necessarily mean that the proteins are expressed at equal amounts.

In the pIVEX MBP Fusion vector map, between MBP and Factor Xa cleavage site, there are 21 additional amino acids (NAAAMHSSSNNNNNNNNNNLG). What is this sequence good for?

The linker that is present in pIVEX MBP Fusion vector is the same that is used in the pMAL plasmid from New England Biolabs. They have also pMAL vectors with other protease cleavage sites (Enterokinase, Genenase I) and the linkers differ slightly. In pIVEX MBP fusion, we added this long Serine-Asparagine linker sequence to MBP to guarantee a good cleavage by factor Xa protease. A shorter linker was analyzed before and revealed negative influence on protease cleavage, probably because of a reduced accessibility of the cleavage site.

Which requirements does RTS have regarding purity of the expression plasmid?

Our recommendation for plasmid preparation is to use midi- or maxiprep protocols, for example our Genopure columns (ion exchange technique). We do not recommend to use Miniprep DNA because the purification method used here (silica) does interfere with RTS and results in reduced yield.
The purity of the DNA can be verified by photometric measurement; the OD260/OD280 ratio should not be lower than 1.7.

No PCR product in RTS E. coli Linear Template Generation Sets found.

This can have one of the following causes:

 

 

Possible Cause	Recommendation
Secondary structures of the primers	a) Try to minimize secondary structure and dimer formation when designing primers b) Raise the primer concentration in the PCR or use longer primers without G or C nucleotides at the 3'-end if a G + C content of 60 % is not feasible.
Inadequate annealing temperature	a) Make sure the right annealing temperature was used for the PCR (5° to 10°C lower than Tm). b) Adjust the annealing temperature to accommodate the primer with the lowest melting temperature.
Concentration of MgCl2 too low	Determine the optimal MgCl2 concentration for each template/primer pair by testing the pair in a reaction series containing from 1 mM to 4.5 mM MgCl2.
Template concentration inadequate	Optimize the concentration of template DNA in the PCR.

A nonspecific amplification in RTS E. coli Linear Template Generation Sets occurred.

This could be due to one of the following causes:

Possible Cause	Recommendation
Low specificity of the primers	a) Make sure that the primers specifically flank the 5'- and 3'-ends of your gene and are not complementary to other sequence regions of the template DNA. If necessary, increase primer length. b) Use hot start techniques. c) Use higher annealing temperature to increase specificity.
Concentration of MgCl2 too high	a) Avoid excessive free magnesium, which leads to nonspecific amplification. b) Determine the optimal Mg2+ concentration by performing a series of amplifications in the presence of 1-4.5 mM MgCl2.

No product in the first PCR of RTS E. coli Linear Template Generation Sets found.

This could be due to one of the following causes:

Possible Cause	Recommendation
The template concentration is too low	Increase concentration of templates.
The primer concentration or sequence is not optimal	a) Verify primer sequences. b) Use high quality primers. c) Optimize primer concentration. Check primer concentrations on agarose gel and try to avoid imbalanced concentrations. d) Check the melting temperature, purity and GC content of the specific primers.
The extension time was too short	Increase extension time to 2 min/kb of PCR target.
The annealing temperature was too high	Lower annealing temperature in 5°C steps.
Insufficient Mg2+ concentrations	Optimize the Mg2+ concentration by testing the reaction in the presence of a range (1.0–4.5 mM) of MgCl2 concentrations.
Multiple contributing factors	a) Test reaction with positive control template and primers of known performance. b) Start over, using freshly made solutions of dNTPs, template and primers. c) Use PCR Optimization Kit (Cat. No. 1 636 138).
The quality of the template is poor (degraded, contaminated, conttaining inhibitors)	a) Prepare new dilution of template. b) Store template at –15° to –25°C in small quantities and avoid repeated freeze/thaw cycles.
GC-rich target	Use GC-RICH PCR System (Cat. No. 2 140 306).

The product band of the first PCR of RTS E. coli Linear Template Generation Sets is not sharp.

The cause for this is a secondary amplification product. Carry out the following steps:

• Check Mg²⁺ concentration and cycle conditions.
• Optimize primer concentration.
• Raise annealing temperature in 3°C steps.
• Decrease number of cycles.
• Decrease concentration of template.

Nonspecific product bands occurred in the first PCR of RTS E. coli Linear Template Generation Sets.

This can be due to one of the following causes:

Possible Cause	Recommendation
Concentration of MgCl2 too high	a) Avoid excessive free Mg2+, which leads to nonspecific amplification. b) Optimize MgCl2 concentration by testing the reaction in the presence of a range (1.0–4.5 mM) of MgCl2 concentrations.
Nonspecific binding of primers	a) Use higher annealing temperature to increase specificity. b) Check and optimize primer concentration. c) Redesign primers to get more specific binding to target and/or to allow a higher annealing temperature. Note: Starting with a plasmid containing a T7 promoter and/or a T7 terminator sequence (e.g., pIVEX, pET, pDEST vectors) the first PCR product has to be purified by agarose gel electrophoresis to prevent contamination of the linear template with the plasmid.

No product occurred in the second PCR of RTS E. coli Linear Template Generation Sets.

This can be due to one of the following causes:

Possible Cause	Recommendation
The template concentration is too low.	Check the concentration of the product of the first PCR on an agarose gel and increase the amount of template in the second PCR.
The extension time was too short.	Recommendation: Increase extension time to 2 min/kb of PCR target.
Insufficient Mg2+ concentrations.	Optimize the Mg2+ concentration by testing the reaction in the presence of a range (1.0-4.5 mM) of MgCl2 concentrations.
Multiple contributing factors.	a) Perform a control reaction. b) Start over, using freshly made solutions of dNTPs, template, DNA and primer dilutions.
The quality of the template is poor (degradation, contamination, containing inhibitors).	a) Prepare new template dilution. b) Store template at -15° to -25°C in small quantities and avoid repeated freeze/ thaw cycles.
GC-rich target.	Use GC-RICH PCR System (Cat. No. 2 140 306).

The product band of the second PCR of RTS E. coli Linear Template Generation Sets is not sharp.

The cause for this is a secondary amplification product. Recommendations:

• Check Mg²⁺ concentration and cycle conditions.

• Decrease number of cycles.

• Decrease concentration of template.

Nonspecific product bands in the second PCR of RTS E. coli Linear Template Generation Sets occurred.

This is due to one of the following causes:

Possible Cause	Recommendation
Nonspecific binding of primers	a) Use higher annealing temperature to increase specificity. b) Either decrease the amount of primers in the first PCR to 0.1 µM or purify the product of the first PCR from an agarose gel. c) Make sure the T7 Promoter and T7 Terminator primers in the second PCR don’t bind to the template DNA from the first PCR.
Approx. 400 bp (His6-tag/HA-tag/AviTag) or 1500 bp (MPB Fusion) by-product	Some gene-specific primers cause a nonspecific by-product to appear in the second PCR, due to primer dimer formation and carry-over contamination (from the first PCR). In this case, either reduce the concentrations of primer and MgCl2 in the first PCR or remove the primer dimers from the first PCR product with the High Pure PCR Product Purification Kit (Cat. No. 1 732 668) or the Agarose Gel DNA Extraction Kit (Cat. No. 1 696 505).

High background of non-recombinants occurs after the transformation of pIVEX vectors.

This could be due to one of the following causes:

<br />

<br />

<table bordercolor="#000000" width="100%" border="1">
<tbody>
<tr>
<td> <font size="2">Possible Cause</td>
<td><font size="2"> Recommendation</td>
</tr>

<tr>
<td><font size="2"> Use of an inappropriate medium. </td>
<td><font size="2"> Make sure that your selected medium contains the correct, active antibiotic by performing a mock transformation reaction without DNA. No colonies should be obtained in this control transformation.</td>
</tr>

<tr>
<td><font size="2"> Incomplete digestion of the vector. </td>
<td><font size="2"> After the first digestion step, purify the vector/insert with the High Pure PCR Product Purification Kit (Cat. No. 1 732 668). Then, perform the second digestion step of the vector/insert in the optimal buffer.</td>
</tr>

<tr>
<td><font size="2"> Unsuccessful dephosphorylation of the vector.</td>
<td><font size="2"> a) <font color="#000000">Perform a control reaction with the vector alone (no insert) to determine the degree of religation. Only a few colonies should be obtained in this reaction.
 b)  <font color="#000000" size="2">Use fresh (shrimp) alkaline phosphatase.
 c) Increase the incubation time.</td>
</tr>

<tr>
<td><font size="2"> Excess of linearized, phosphorylated vectors. </td>
<td><font size="2"> Depending on background, reduce the amount of linearized vector in the ligation reaction two- to fivefold. <b><i><font face="Arial, Helvetica" color="#000000" size="2">Note:</i></b> If the ratio of vector to insert is too high or too low, religation is favored.</td>
</tr>
</tbody>
</table>

No control protein is visible after expression in RTS.

This could be due to one of the following causes:

• Contamination with RNases. Recommendation: Repeat the experiment and be careful to work RNase-free at each step.
• Air bubbles in the large feeding chamber of the RTS 500 device. Recommendation: Remove any air bubbles from the feeding chamber before starting a run.
• A kit component is inactive or degraded. Recommendation:
  Contact RiNA GmbH technical service.
• A kit was expired. Recommendation: Order a new kit.
• The kit has not been stored at -20°C. Recommendation: Order a new kit.

No target protein is visible but the control protein is expressed normal.

This may be due to one of the following causes:

Possible Cause	Recommendation
The protein concentration is too low	Load the maximum amount of sample on the gel.
The protein is insoluble.	Analyze supernatant and pellet.
The tag is hidden.	a) Try tagging the protein in a different position. b) Try protein specific antibody. c) If the protein is His6-tagged, try a different anti-Histidine antibody (e.g. anti-Penta-Histidine antibody). d) Check the protein on a Coomassie stained gel in addition to the Western blot.
Cloning error	Check the sequence.
Impure plasmid DNA	a) Make sure that the 260 nm/280 nm absorbance ratio is at least 1.7. b) Perform a phenol extraction if purity is low. c) Make a new plasmid preparation.
No initiation of translation due to strong secondary structures of the mRNA	a) Use ProteoExpert (Cat. No. 3 115 569 for one access number, Cat. No. 3 115 577 for five access numbers, Cat. No. 3 115 585 for 25 access numbers) to optimize the template sequence. b) Clone the gene into different pIVEX-vectors to prepare a new expression construct. c) Try expressing the target as an N-terminally tagged fusion protein in pIVEX MBP Fusion Vector (Cat. No. 3 268 985).
The expressed protein interferes with the translation or transcription process.	Express the control protein in the presence of the target protein. If control protein expression is inhibited, the target protein can not be expressed in RTS.

Is it possible to express His6-tagged proteins with the RTS AviTag Biotinylation Kit, Plasmid or RTS AviTag E. coli Biotinylation Kit, Linear Template?

The vectors and Linear Template Generation Set provided with the RTS AviTag Biotinylation Kits do not encode for a His6-tag. It is, however, possible to attach a tag sequence to the target gene, e.g. by PCR mutagenesis, prior to inserting this gene into the AviTag vector/Linear Template and thus express biotinylated proteins with an additional affinity tag.

As from your experience, which are the common causes for low yield?

Which role has DNA purity of templates?
Is the quality obtained by plasmid minipreparation enough or do you generally recommend eg a phenol-chloroform extraction?
Are there other possibilities?

 

 

Low yield may be due to one of the following causes:

Possible Cause	Recommendation
The expression time is too short	Extend expression time.
Contamination with RNases	Repeat the experiment and be careful to work RNase-free at each step.
The tag has a negative influence on the folding of the protein	a) Try preparing a different expression construct by cloning the gene into different pIVEX vectors. b) Introduce different epitope tag sequences via PCR. (For example, use RTS E.coli Linear Template Generation Sets: Cat. No. 3 186 237 for His6-tag, Cat. No. 3 315 860 for HA-tag, Cat. No. 3 258 828 for MBP Fusion, Cat. No. 3 521 818 for AviTag.)www.proteoexpert.com
Low initiation of translation due to strong secondary structures of the mRNA	Use ProteoExpert to optimize template structure and make a good yield more probable.
The amount of template DNA is not optimal	Vary (increase) the DNA concentration to get optimum results.

To be on the safe side I would use maxiprep DNA (although miniprep generally also works). Phenol-chloroform is a little bit over the top or even dangerous if not done extremely carefully, since traces of organic solvents will inhibit in vitro translation. For the same reason DNA isolated out of agarose gels should not be used.

Sufficient protein expression, but low yield of active protein.

This can be due to one of the following causes:

Possible Cause	Recommendation
Dependence on cofactors	Add necessary cofactors.
Requirement for disulfide bonds	a) Proteins with more than three disulfide bonds may only be expressed in a functional form in the RTS E. coli system by using the RTS 100 Disulfide Kit or the RTS 500 Disulfide Kit. These kits based on E. coli lysate are especially for the expression of disulfide bonded proteins from plasmid templates. b) For proteins with up to three disulfide bonds: After synthesis, allow oxidation to promote formation of disulfide bonds. (See e.g., Ahmed et al., 1975 or Odorzinsky and Light, 1979.)
Dependence on secondary modifications	The E. coli lysate cannot introduce posttranslational modifications such as glycosylation, phosphorylation, or cleavage of a signal sequence.
Dependence on chaperones for correct folding	a) Add chaperones (see Rudolph et al., 1997). b) Use RTS GroE Supplement (Cat. No. 03 263 690 001). It can fold small (20-60 kD) proteins.

What is the protein yield of the different RTS Kits?

The purity of the template is important for the reaction.

You should always check it. The absorbance ratio 260 nm/280 nm should be at least 1.7. If not, you have to perform a phenol extraction. If this does not help, a new plasmid purification is needed.

The protein yield of the different kits could be find bellow:
The amount of template DNA has no significant influence in the concentration range we recommend. If it is too high, the reaction can be inhibited

RTS 100 E.coli HY Kit: up to 25 µg/50µl reaction
RTS 500 E.coli HY Kit: up to 5 mg /ml reaction
RTS 500 ProteoMaster E.coli HY Kit: up to 5 mg/ml reaction
RTS 9000 E.coli HY Kit: up to 50 mg /10 ml reaction

What is the success rate of RTS?

The success rate of RTS is more than 85% for all proteins expressed.
However, this does not guarantee positive results for any particular protein.
The success rate of expression increases dramatically if different tags and tag positions are used or the sequence is optimized for expression using ProteoExpert

Following the hints given in the RTS Troubleshooting Guide may also improve the success. For yields that can be expected with the different kits please compare Protein Yield of different Kits.

What is the maximum size of proteins that can be expressed in RTS?

Proteins in the molecular weight range from 5 to 120 kD have been successfully synthesized. It is expected to be problematic to express proteins of a size larger than 200 kD, also because cloning of the gene will become difficult then. For small proteins please keep in mind that the semi-permeable membrane that separates the feeding compartment from the reaction compartment has a 10 kD cut-off. For examples please refer to the list of expressed proteins on our special interest site.

Who is RiNA GmbH

RiNA was founded as a spin-off from the Institute of Biochemistry (Prof. Dr. Volker A. Erdmann), Free University of Berlin. RiNA successfully provided major Research & Development in the field of prokaryotic and eukaryotic protein expression systems.'Roche, RiNA GmbH, Berlin, and 5 PRIME GmbH, Hamburg agreed that effective September 15, 2009 the complete RTS Protein Expression portfolio of Roche Applied Science will be transferred to RiNA GmbH and 5 PRIME GmbH.

RiNA will contribute its longstanding experience in the field of cell-free expression systems and continue production at the same quality standards defined by ROCHE.

Is it possible to express non-E.coli proteins?

Normally it is not necessary to adapt the reaction mix to a certain codon usage because all tRNA species are present in excess. If a phage protein turns out to be poorly expressed, it's most likely not because of codon usage but because of some physiological interference with the lysate.

For your further information:
RiNA GmbH offers a large selection of products for cell-free protein expression.

www.rina-gmbh.eu

Is it possible to express eukaryotic, viral and plant proteins in RTS?

Yes. To ensure expression of non-E.coli proteins and overcome differences in codon usage, an excess of tRNAs has been supplemented into the lysate.

Is it possible to express membrane proteins?

Membrane associated proteins as well as transmembrane proteins have been expressed successfully (e.g. TIM17 and TIM23). However, transmembrane proteins tend to be insoluble. Optimization with detergents is possible.

Is it possible to express toxic proteins in RTS?

In prinicple it is possible to express toxic proteins, as long as they do not interfere with the transcription/translation machinery. We already have a few examples for the expression of proteins which could not be expressed in E.coli.
Expression of cell toxic protein MID 1 using the RTS 500 E. coli Circular Template Kit

Amplification of pIVEX plasmids with a cloned toxic gene is no problem in XL1Blue. The pIVEX plasmids contain a T7 promoter for expression. The gene is only translated if a T7 RNA polymerase is present in the host cells. This is not the case in XL1 Blue. In these cells, the protein can not be expressed and can not exert its toxic effect on the host cell.

Does RTS allow expression of biologically active proteins?

RTS has already been used to express many functionally active proteins, e.g. h-Interleukin-2, GFP, b-Galactosidase and single-chain antibodies. Please refer to the list of expressed proteins and the literature cited on our special interest site.

Can proteins with multiple subunits expressed in RTS?

Yes. Vectors coding the sequences for different subunits can be added into one reaction for coexpression. We have an Application Note describing one example for the expression of a large homo-multimeric enzymatic protein; beta-Galactosidase.

Can proteins be coexpressed in RTS?

Yes. One example is given in the Results section in our RTS Special Interest Site:
Co-expression of GFP and CAT using the RTS 500 E. coli Circular Template Kit.

Coexpression of two (or more) proteins using RTS leads to more flexibility and to more accurate production of the desired relative amounts of protein when compared with cellular coexpression. In cellular systems there is no strict control about the copy number of each plasmid in transfected cells and consequently about the amount of each protein. The use of the same amount of plasmid sometimes leads to different amounts of expressed protein. Using RTS, the concentration of plasmid can be adapted to obtain the desired amount of both proteins. The recommendation is to use 0,5 µg of each plasmid for coexpression of two proteins.

Can proteins be labeled in RTS?

Yes. Labeled amino acids (radiolabeled or heavy atom-labeled) can be incorporated into proteins expressed with RTS. A mixture of all amino acids except methionine is provided in one tube and methionine is provided in a separate tube as components of the HY kits. Researchers can add labeled methionine and expect highly specific labeling of the product because of this kit configuration.
The RTS Amino Acid Sampler provides all amino acids separately, thus giving more freedom of choice as to which amino acids can be modified in a certain protein.  

Pack insert

Labeling in RTS has several advantages:
1.Only the template DNA is expressed in RTS. Therefore, only the protein of interest is labeled.
2. No metabolic scrambling occurs. Therefore, labeling with single amino acids is highly specific.

Can proteins expressed in the RTS be used for NMR spectroscopy or X-ray crystallography?

Yes; e.g. Phosphoserine Phosphatase. In vitro protein production for structure determination with the RTS) has been successfully expressed in RTS and used for structure determination. An overview is given in the RTS Application Note :Protein Labeling.

How can proteolytic cleavage be avoided?

Although not required, it is possible to add protease inhibitors, but they must be EDTA free (for example Complete Protease Inhibitor Cocktail Tablets, without EDTA).

What is the most reliable way to analyze protein expression?

We recommend Western blotting, and/or a functional assay.The RTS Application Note Protein Analysis and Purification includes a protocol for Western blotting with the description for colorimetric and chemiluminescent detection.

Which vectors can be used for expression in RTS in addition to the pIVEX vectors?

The pIVEX vectors have been specifically designed and optimised to work in RTS. Any vector to be used in combination with RTS must include the following elements: Circular closed form Target gene under control of a T7 promoter Ribosomal Binding Site (RBS) The distance between the T7 promoter and the start codon (ATG) should not exceed 100 base pairs. The distance between the RBS and the ATG should be in the range of 5-8 base pairs and should be AT-rich. A terminator sequence at the 3 ' end of the gene is optional, but recommended. An overview of other expression vectors tested in RTS is given in a Biochemica article. Alternatively, linear templates can be generated using the RTS Linear Template Generation Sets for expression in the RTS 100 E. coli HY Kit. For scale-up, these linear templates can be cloned in any PCR cloning vector. Cloning procedures to convert linear to circular templates are described in the RTS Application Note. From linear to circular expression templates.

Is the E.coli polymerase blocked?

Yes, only transcription via the T7-polymerase can work in the RTS lysate.

How can I detect aggregated protein?

Take 15 µl of the reaction mixture and centrifuge it for 5 min. Pipet off the supernatant and dissolve the pellet in 15 µl of SDS sample application buffer. Run SDS-PAGE with the supernatant and the pellet side by side, and detect by Western Blot.

Can I use the device again?

We do not recommend to use the device again for the following reasons:
Fouling of the device can decrease the yield significantly.    
RNase contamination can lead to no protein yield.

How can I optimize protein expression with RTS?

Many factors can lead to a low expression level of the protein, including the expression yield or solubility. But it can also be due to the detection method. Here are some suggestions for improving the results:

- vary the amount of expression plasmid.
- vary reaction time.
- use chaperones for the correct folding of proteins between 20 and 60 kDa.
- add detergents for hydrophobic proteins. Unfortunately there is no way to predict which detergent will be successful.
- add cofactors.
- try different tags and tag positions. MBP-fusion proteins are often very soluble. The fusion vector contains a Factor Xa cleavage site that allows you to subsequently purify your protein away from the MBP (Maltose Binding Protein).
- vary reaction time.
- vary reaction temperature (+20 to +37&#176;C). The higher your reaction temperature, the more likely it is that aggregated proteins will be obtained.

For your further information: RiNA GmbH offers a large selection of products for cell-free protein expression. Rapid Translation System is a scalable in vitro transcription/translation protein expression platform, that produces sufficient amounts of protein for characterization studies, functional assays, or structural analysis. Two modular systems are available: the prokaryotic E. coli or the eukaryotic Wheat Germ. For a complete overview, please visit and bookmark our ""Rapid Translation System"" Special Interest Internet Site.

Please also note our Printed Materials.

How pure will the protein be if expressed in RTS?

The protein will not be pure, as it is expressed in an E.coli lysate. The background protein concentration is approximately 10 mg/ml.

Which purification methods can be used?

Depending on the tag used different purification protocols can be applied. Please refer to our RTS Application Note Protein Analysis and Purification for protocols for the purification of a His6-tagged, a HA-tagged and a MBP fusion protein.

Can I directly use proteins expressed in RTS for whole-animal studies?

Proteins should first be purified. A substantial level of endotoxin is present in the E.coli lysate.

What is ProteoExpert ?

ProteoExpert is...
- a web-based service application for rational and efficient optimization of protein expression templates.
- a bioinformatics tool enabling you to maximize protein yields. The current version ProteoExpert RTS E.coli HY is specially adapted for protein synthesis using the cell-free Rapid Translation System (RTS).