Sequence errors in your gene for expression Part II follows on from an earlier blog where we discussed a problem we were having producing a particular protein after synthesizing a gene based on very old sequence data. The baculovirus we made containing this synthetic gene produced very low levels of recombinant protein that were hardly visible after immunoblot analysis. This wasn’t a good start to a project where a purified protein was required for diagnostic purposes.
We went on to discuss possible sequence errors in the target gene as a possible reason why we were not seeing much protein production. Our suspicions were accentuated because earlier reports where a cDNA was used for expression purposes had yielded good levels of recombinant material. Unfortunately, we didn’t have access to this cDNA clone to enable us to remake the virus for protein production.
We resorted to comparing more recent data for our target gene with the information we had downloaded from GenBank originally. This highlighted an alarming number of inconsistencies between the sequences. In consequence, we designed a new synthetic gene, had it made and then inserted it into one of our flashBAC™ vectors. A few days later, having made our recombinant virus and done some quick tests for protein expression, we were very pleased to see a nice band reacting with our specific antiserum on immuno blots. The next steps are to scale up protein production and purify the protein, which unfortunately we can’t identify in this blog.
The lesson we have learned from this experience is never assume GenBank sequence is 100% accurate! Although our second round of gene synthesis also used a GenBank file, we were able to be reasonably sure it was accurate owing to the fact that many other independent data entries supported it. This still left a slight uncertainty in the outcome of the project but as anyone doing protein expression will testify, there is nothing like a shot of adrenalin in the afternoon as you wait for your western blot to develop bands of recombinant protein!
If you are having a particular problem with producing your recombinant protein send us an email via the OET Ltd website with some general information about your project and we will attempt to help.