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Dear Reza
If I am correct, when you optimize a gene with a company, normally they optimize two things: they change sequence with silent mutation to minimize
RNA secondary structures and they change also the sequence to adapt to the host’s codon usage according to tables (see website mentioned by Mark)
The first optimization, RNA secondary structures, is independent of the host organism and probably it may be the most important optimization in a number of genes, explaining why e coli optimization is good for insect cells.
For codon usage, what could be worth is to check if you have in your sequence several rare codons following each other ( using the tables mentioned).
If yes, it could explain some problem of expression. Repetition of rare codons could be an issue
Hope it could help
JB
Laboratory of Structural Biology and Radiobiology
I2BC (Institute of Integrated Biology of the Cell)
iBiTec-S (Institute of Biology and Technologies of Saclay)
CEA, Saclay, Bât 144, room 140D,
91191 Gif-s-Yvette, FRANCE
phone: +33 (0)1 69 08 76 77
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Envoyé : mardi 16 février 2016 15:44
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Objet : Re: [ccp4bb] Protein expression in insect cell
Hi Reza,
We have also used E. coli-optimized constructs in insect cell systems with no problems. It might worth checking a few different insect cell lines. We have a case when little expression is observed in Sf9 and Sf21, but massive amount (50
mg/L secreted) is obtained from High Five (same as T. ni from other companies). I have heard that screening a few different media is a good idea, but I have not experience this problem first hand. PCR the virus directly to see if the expression cassette is
still there is probably also a good idea.
Best,
Pun
On Tue, Feb 16, 2016 at 8:47 PM, Reza Khayat <[log in to unmask]> wrote:
Hi,
We have an E. coli codon optimized gene that we would like to try expressing in insect cells. The protein is not expressing and we are concerned that it may be because
of the codon optimization. Could this be a possibility, or are the insect optimized codons similar to that of E. coli? Thanks.
Best wishes,
Reza
Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031