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Gene to Protein Webinar Questions and Answers
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You and your colleagues submitted interesting questions during the webinar, “From gene to protein—extending gene synthesis to reliable protein expression,” so we’ve collected and answered them here. Click on a question to see the answer.
Q: How effective is your gene optimization in comparison to the competitors’ software?
Q: How fast can you deliver the protein if I send you a gene sequence?
Q: Why do you differentiate between HEK and CHO? Which system is superior in terms of expression?
Q: Can you enter your own codon usage table for gene optimization on the GeneArt system?
Q: What is the link for the paper you mentioned? (Paper on mammalian expression optimization study)
Q: What about optimization of a mammalian protein for E.coli expression?
Q: Is there any published paper related to your optimization algorithm?
Q: Could you optimize bacteria gene which will be expressed in plant (rice)?
Q: Can I use gene design and synthesis for expression in wheat germ-based cell-free system?
Q: Where can I find more information on your 'Sliding Window' algorithm?
Q: How effective is your gene optimization in comparison to the competitors’ software?
A: Very effective! For example we have tested our mammalian expression optimization and published the data. Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression Fath et al., 2011 PLoS ONE
Our optimization was successful in an autologous setting, and we observed up to a 25-fold protein expression increase. We found that 96% of all tested proteins expressed equal to or better than the wild-type proteins.
In addition, we performed internal comparison studies with genes optimized and synthesized by competitors and found the GeneOptimizer™ software outperformed competitor-optimized genes in each case. Our company guidelines do not allow publishing these data, but you are welcome to test us against the competition!
Q: Does gene optimization work with every protein, e.g., can I also get more protein if I already have a great expression?
A: Yes, we know from our own data and from customer feedback that optimization can improve even well-expressed proteins. For other expression systems, we have not systematically checked that, and it is different from protein to protein, but for example for antibodies 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 this is improved, not even with a combination of optimization and our best expression system (Expi293™ System).
Q: How fast can you deliver the protein if I send you a gene sequence?
A: It depends on the current workload in the lab, the number of ordered proteins, and complexity to purify, but we can typically deliver within 30 business days starting from gene synthesis. If we encounter any problems during gene synthesis, cloning, protein expression or purification, we will contact you.
Q: Why do you differentiate between HEK and CHO? Which system is superior in terms of expression?
A: In our hands HEK293 cells usually express more than twice the amount of protein, but this is also dependent on the protein. Some customers do prefer the human cells, others CHO cells.
Q: What happens if my protein is toxic or does not express? How do you guarantee that I get something for my money?
A: We usually set up the project in milestones and in cases where we have to stop at a specific milestone, we only charge for the work we have done.
Q: Can you optimize a genetic sequence for dual organism expression? For example, in cases 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. It depends on the organisms you choose for expression, since their respective codon usages have to be compatible. Accordingly, dual optimization for E. coli and yeast expression would not be recommended because the most preferred codons in E. coli are the more rarely used codons in yeast, and vice versa. The same would be true for E. coli and mammalian expression. On the other hand, more similar expression host pairs, such as Pichia and Saccharomyces, or human and hamster (CHO), will work very well for dual expression optimization. It clearly depends on each individual case, and we will advise you on the best solution for your project.
Q: Can you enter your own codon usage table for gene optimization on the GeneArt system?
A: Yes, you can submit your own codon usage table to us. Please contact GeneArtSupport@lifetech.com, and the support team will send you an Excel spreadsheet where you can enter the desired CUT, and we will optimize your genes according to that CUT. It’s not possible to upload your own CUT to the online portal.
Q: For a 1,000 bp gene how long do you need to optimize, synthesize and subclone in an expression vector?
A: The fastest way for obtaining an expression-ready clone is our GeneArt Gene Synthesis plus Express cloning service. For genes up to 1,200 bp (GC content 10–80%) we have a standard production time for gene synthesis and express cloning of 11 business days with speed upgrades available. See the GeneArt website for more information.
Q: What is the link for the paper you mentioned? (Paper on mammalian expression optimization study)
A: Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression Fath et al., 2011 PLOS one
Q: What about optimization of a mammalian protein for E.coli expression?
A: If a gene is optimized for expression in any host it is of no concern where that gene/protein came from. So, yes, mammalian proteins can very well be optimized for expression in E.coli.
Q: Is there any published paper related to your optimization algorithm?
A: Please refer to: The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Raab et al., 2010 Systems and synthetic biology
Q: .: If you are overexpressing a small soluble protein in E. coli and it seems to have a high tendency to form inclusion bodies what approaches can you take to prevent this? I want to isolate "native" protein that means I want to prevent inclusion bodies forming. I have tried lower [IPTG] and lower temperature, are there any other approaches to take during growth?
A: If the protein is a mammalian protein you might, of course, consider expression in mammalian cells. We also offer this as a service. If E. coli is a must, you may also try different expression vectors with either varying promoter strength or plasmid copy number. A de-optimization can also help reducing the expression strength, thereby the tendency to form inclusion bodies. Denaturing and refolding sometimes works as well. Then there is also the option to add tags which sometimes help solubilizing (MBP, GST) or to generate truncation variants which may be more soluble while still retaining phenotype.
Q: Do you have a protein production/purification system that when I receive the expression vector I could then use it to express and purify myself in E. coli?
A: Yes, just visit our website for E.coli expression systems. You can order an E. coli optimized gene and use this for your E. coli expression.
Q: Could you optimize bacteria gene which will be expressed in plant (rice)?
A: Yes, definitely. Our online order portal offers several mono- and dicot. plant hosts to optimize your gene. Rice is amongst them.
Q: Can I use gene design and synthesis for expression in wheat germ-based cell-free system?
A: Yes. In this case use the optimization functionality of the online order portal and optimize your gene for wheat.
Q: According to insuline synthesis for diabetic people, modern approaches as far as I know use bacteria for wider production of insuline instead of mammals to avoid the cost, small production and allergies.. So isn’t it more preferable either way to avoid mammalian protein production for pharmaceutical proteins?
A: It depends on the protein, some proteins cannot efficiently be produced in E.coli others require post translational modifications for full functionality. All antibody drugs, for example, are manufactured in mammalian cell lines
Q: Where can I find more information on your 'Sliding Window' algorithm?
A: You can find more information in the related publication: The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Raab et al., 2010 Systems and synthetic biology
Q: Did I get it right that your Express cloning service can reduce the delivery time of cloned genes by 5 days?
A: Yes, you'll receive a gene up to 1.2 kb cloned in selected expression vectors in typically 9-11 business days. That is up to 5 days less than if ordered as classical subcloning after gene synthesis. Express cloning can simply be added to the gene synthesis order when you are ordering via the GeneArt portal
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