HIS tagged protein from baculo

Darren A. Natale dnatale at box-d.nih.gov.nospam
Thu Feb 5 08:41:27 EST 1998


un691cs at genius.embnet.dkfz-heidelberg.de wrote:

> we have cloned a HIS-tagged protein in baculovirus. It
> is a novel TGF-beta member, and dimeric. We get
> some expression; the protein is indeed dimeric and looks o.k.
> in a western. We can purify it with the his-tag.
> However, we need at least 100 micrograms
> for our initial assays, and this doesn't seem to work.
> 
> question 1: what methods can we use to determine the amount of
> protein. It is very dilute. We have tried Bradford, but aren't
> in the linear range. Any pointer welcome here. I just want to
> know more or less exactly how much protein we have.
> 
> question 2: how do I optimize my production of the protein ?
> what parameters to watch ? How much protein is normal, how much
> can YOU make. any tricks not found in the manuals ? Are certain
> proteins toxic to baculo ? Insect cells also produce TGF-betas.

I presume you are using spinner flasks?  I found out the hard way
that you have to keep the volume lower than you may expect given the
volume size of the flask.  For example, when I tried expressing a
protein in Sf9 using a 1 L culture in a 1 L flask, I got nothing!  If
I do the same protein, same cells, same flask, but drop the culture
volume down to 400 mL, it works fine.  Bear in mind that I was relying
on the spinner to provide aeration.  I think this problem can be avoided
by bubbling air through the liquid, but I never tried it that way 
(I don't have the equipment).

I have also seen reference to problems if the MOI is too high.  There
tends to be some degradation and/or low expression.

D. Natale
(remove ".nospam" from address to reply by mail)

> 
> thanks, Clemens Suter-Crazzolara, Heidelberg



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