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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?
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!
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).
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.
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.
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.
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.
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.
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.
A: Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression Fath et al., 2011 PLOS one
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.
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
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.
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.
A: Yes, definitely. Our online order portal offers several mono- and dicot. plant hosts to optimize your gene. Rice is amongst them.
A: Yes. In this case use the optimization functionality of the online order portal and optimize your gene for wheat.
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
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
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
For Research Use Only. Not for use in diagnostic procedures.