expression optimized gene webinar

Your colleagues submitted interesting questions during the webinar, “Combine expression-optimized genes with an advanced expression system to improve your results,” so we’ve collected and answered them here.

Find the answer quickly by clicking your desired question.

Q- Did you consider the cis-element which increase the protein expression when you optimize the condon?

Q- If I already have the gene, and got it from genart, I guess protein purification will be cheaper, isn't it?

Q- The webinar will be online another day? I lost the first part of the presentation.

Q- What expression systems do you offer to codon optimise for? Do you codon optimise for expression in Schneider-2 Drosophila cells?

Q- What if you wanted to optimize a genetic sequence for dual organism expression? For example, in the case where you're trying to have an optimal expression in either yeast or e coli and want a sequence that will work well in both.

Q- What type of transfection do you perform, targeted to a specific or random integration?

Q- What is the usual price range for a gene optimization service?

Q- You may have said this and I missed it, but is the user able to enter their own codon usage table for gene optimization on the LifeTech GeneArt system?

Q- Does gene expression optimization work with every protein, so can I always better yields if I already have a good expression?

Q- How fast can you deliver a protein if I send you the gene sequence?

Q- Do you send the expression plasmid you use for transfection?

Q- Why do you differentiate between HEK293 and CHO cells what cells are superior in terms of expression?

Q- What happens if my protein does not express at all, how do you guarantee that I get something for my money?

Q- Do you check for biosecurity?

Q- I have query regarding His tagged protein purification while using Ni-NTA column I am getting nonspecific protein band on SDS-PAGE --3 band high molecular weight & 1 low molecular weight (27KD). I have changed washing conditions, resin column but still getting the same problem. I came to know that these are endogenous proteins in the host that we are using. How to get rid from these non specific protein band while purification?

 

Q-Did you consider the cis-element which increase the protein expression when you optimize the condon?

A- We only optimize the respective open reading frames of your desired gene. We remove all regulatory elements wherever possible in order to avoid alternative transcription start sites. Negative cis-acting sites (such as splice sites, poly(A) signals, TATA-boxes, etc.) which may negatively influence expression will be eliminated as well. We do not optimize promotor regions or else.

Q- If I already have the gene, and got it from genart, I guess protein purification will be cheaper, isn't it?

A- Yes, if we produced the gene already, we have a retain sample inhouse that we can use for plasmid preparation supporting the transient transfection for protein expression. So you do not need to send us anything and we will not charge the gene synthesis. However, please make sure we optimized the gene for the organism you want us to use for expression, e.g. we do not recommend to use an E.coli optimized gene for protein expression in mammalian cells.

Q- The webinar will be online another day? I lost the first part of the presentation?

A- Yes, as a registered participant you can access the webinar at any time. A link to the online availability of the webinar will be sent to you right after the presentation and the webinar will be available "on demand" on the Life Technologies Synthetic Biology website in short time (Synthetic Biology Webinars)

 

Q- What expression systems do you offer to codon optimise for? Do you codon optimise for expression in Schneider-2 Drosophila cells?

A- Yes - we do routinely optimize according to the CUT of Drosophila cells, which is working very well for Schneider-2 cells. We can optimize to all CUT which are available and we certainly do, to all standard expression hosts. If you want to send us a special codon usage table for your cells, please contact the service team @GeneArtSupport@lifetech.com


Q- What if you wanted to optimize a genetic sequence for dual organism expression? For example, in the case where you're trying to have an optimal expression in either yeast or e coli and want a sequence that will work well in both?

A- Dual optimization does not make sense in every case but depends on the organisms you choose for expression, since the respective codon usage tables (CUT) have to fit together. Accordingly dual optimization for E.coli and Yeast expression will not be recommended because the most preferred codons in E.coli are more rarely used codons in Yeast and vice versa. The same would be true for E.coli or mammalian expression as demonstrated during the webinar. On the other hand more comparable expression hosts such as Pichia and Saccaromyces or Human and hamster (CHO) certainly will work very well for dual expression optimization. It clearly depends on each individual case and we certainly will advise you on the best solution for your project.

 

Q- What type of transfection do you perform, targeted to a specific or random integration?

A-For standard protein expression services we perform transient transfections since our advanced protein expression systems (Freestyle and Expi) are optimized for this technology. In addition this will assure a much more predictable outcome of your projects with regard to yield and timing. Nevertheless we also offer different cell line geneartion services where e.g. we can provide you with a cell line, expressing your transgene after targeted integration using Life Technologies Flp-In technology, or else.

 

Q- What is the usual price range for a gene optimization service?

A- The gene optimization itself is free of charge, if you order a gene and use our online portal for optimization, you can decide if you want it synthesized as an optimized or wildtype gene. We do not routinely offer gene optimization alone without gene synthesis.

Q- You may have said this and I missed it, but is the user able to enter their own codon usage table for gene optimization on the LifeTech GeneArt system?

A- Yes - you are able to submit your own codon usage table to us ! Please contact GeneArtSupport@lifetech.com; the support team will send you an Excel sheet where you can enter the desired CUT and we will optimize your genes according to that CUT.

Q- Does gene expression optimization work with every protein, so can I always better yields if I already have a good expression?

A- We know from our own data and from customer feedback that optimization can improve even well expressing proteins. This is for sure different from protein to protein, but for example for antibody expression in 293 and CHO cells, we have seen large improvements, even if the antibody is already well expressed. However, if a protein does not express at all, we cannot guarantee that our optimization can change this, but it is definitely worth trying.

Q- How fast can you deliver a protein if I send you the gene sequence?

A- As shown in the presentation, we can go from gene to protein in 30 business days.But the actual production time depends on several paramenters, e.g. the gene length, the number of proteins you order in parallel, the actual workload of the lab and so on. You receive a more accurate due date if we confirm start of the project and in case we encounter any problems during gene synthesis, cloning, protein expression & purification we will contact you.

Q- Do you send the expression plasmid you use for transfection?

A- Yes, we always send the expression plasmid and the purified protein.


Q- Why do you differentiate between HEK293 and CHO cells what cells are superior in terms of expression?

A- I think it is common sense that 293 cells often express more protein. But this can be different depending on the protein. If you want to be sure, you should try. We have customers preferring the human cells, others CHO cells because e.g. due to a specific desired glycosylation pattern.

Q- What happens if my protein does not express at all, how do you guarantee that I get something for my money?

A- We usually set up the projects in milestones and in case we have to stop the project before, we charge only for the work we have done.

Q- Do you check for biosecurity?

A- Yes, we do a biosafety and biosecurity check for every gene we produce. That means we screen every sequence against a database for indexed sequences. If we find a hit, we may have to do a BAFA application at the german authorities to be able to produce the gene. In that case the customer will be informed that this application is needed and that it may take several weeks before the application is granted. In rare cases we take the right to refuse the order due to a biosecurity issue.

Q- I have query regarding His tagged protein purification while using Ni-NTA column I am getting nonspecific protein band on SDS-PAGE --3 band high molecular weight & 1 low molecular weight (27KD). I have changed washing conditions, resin column but still getting the same problem. I came to know that these are endogenous proteins in the host that we are using. How to get rid from these non specific protein band while purification?

A- Co-purification of endogenous proteins containing clusters of Histidins is a quite common observation using a one-step-Ni-NTA purification strategy, especially when you are purifying cellular proteins. In order to get rid of non-specific bound proteins you can 1) increase your production levels in order to saturate the column with your target, 2) use high salt washing conditions to get rid of target bound protein, 3) but presumably you need to follow a multi-step purification strategy and include additional ion exchange chromatography or size-exclusion cromatography steps. For several difficult proteins we already fused a secretion leader peptide to a transgene by Gene Synthesis, in order to succesfuly purify the secreted protein at highest purity from conditioned media (where applicable).