Nature Methods - 3, 1021 - 1032 (2006)

doi:10.1038/nmeth983

You can try this method even if you have different setup for baculovirus

You could try Hi5 cells, I have found that they are often much better for some proteins.

Generally for a large protein of 230kDa you will expect to get weaker baculovirus expression than for a smaller protein. Early promoter expression will not help with abundance of the target because early promoters are much weaker than polh promoters. I recommend you co-infect with AB Vector's ER3 helper baculovirus. It will supply PDI (protein disulfide isomoerase) and human calreticulin during the late expression of the target protein. Their helper virus will be perfectly compatible with Bac-to-Bac in Sf9 cells and you won't have reclone into a transfer vector..

The whole insect/baculovirus system at C-PERL in the USA where I work has been applied to express large proteins that cannot be made successfully in cell culture. Algenex in Europe has a whole insect expression service.

Hi everybody,

Thanks for all of these valuable suggestions.

- To Sandeep Talapatra and Olga Lihoradova : I was very probably infecting my cells with too much baculoviruses, wich is likely to produce defective viruses. I will follow the recommandations given in the Nature Methods given by Sandeep to try to avoid this issue.

- To Olga Lihoradova and Jeffrey Slack : If the first point isn't sufficient to get rid of my production problem, I will try to use the AB verctor's helper baculovirus as advised by both of you.

- To Olga Lihoradova : No, this isn't a secreted protein but a cytosolic one.

- To Efstratios Stratikos : I don't have HI-5 cells in my lab, so I will test it later if my problem persists.

Once again, thank you all, I will tell you how it works in a few...

Hi Yann-Vai,

Couple of comments, you could try making a recombinant baculovirus with either Baculogold triple cut linear DNA or fashBac DNA using Cellfectin II for the transfection, and then use 100 uL of the T-25 transfection supernatant to infect 3 x 106 Sf9 cells in a T-75 flask for 7-12 days until >95% of the cells have lysed. That will give you lots of infectious virus, shaker flasks cultures generate lots of defective viruses. And I know someone who had lots of problems with the Bac-to-bac system for a large protein, which was solved when going to the above. Then do a serial dilution of the baculovirus in 12 to24 well plates with Sf9 cells to find the right MOI for your protein's expression. Once you have found that scale up. The optimal MOI can vary from one protein to another. The problem you will always have is that a protein of 230 kDa is not going to express well compared to a 32 kDa protein just based on the amount of amino acids needed for each protein (7x more aa for a 230 kDa protein). Media can also be a factor, as can FBS if you are using it, I have gone to serum free media because of FBS issues.

Let us know how it goes.

I would follow Jeffrey Slack's advice...very technical and personally that's what I would do for expressing anything greater than 200 kDa in baculovirus at our core facility as well.

Cheers

for secretory expression, we always use the pACGP67 system from BD. it is based on cotransfection using a transfer vector and linear baculoviral DNA. we have secreted upto 210 kDa proteins on a 100L scale using this system. if BD is stopping selling the linear DNA, try to buy Linear Baculoviral DNA from Expression Systems. that works well as well.