Eric Campeau wrote:
>I am currently starting expression in yeast and the manufacturer (of the >expression system) is highly recommending baffled flasks.
>However, we do not have those flasks, only regular Erlenmeyer. Should I follow >blindly the manufacturer's instructions ? If not, is there
>a place I could find some good and cheap baffled flasks (from 250 ml to 2 l)
Variables like vessel size and geometry and the type of shaking or aeration
can be extremely important. In the case of secreted, soluble Kex2 expression,
active enzyme accumulated to >25 mg/l when cultures were grown in 10 ml volumes
in the roller drum but enzyme activity peaks early and is unstable in shake
flasks of any size. Many cases of protein expression benefit from baffled
flasks and rapid shaking but there are exceptional cases that benefit from
slow shaking or aeration without shaking. The composition of media is also
something you should attempt to optimize. Again, in the case of Kex2,
expression was improved with addition of casamino acids and binding to a
Q Sepharose column was improved by lowering the sulfate in the medium.
The best advice is to optimize your assay for protein (hopefully enzyme)
expression and then vary many conditions in growth conditions for your protein.
Try to borrow flasks from folks until you know what is best for your case.
Rosenstiel Center, rm 650 phone (617) 736-4908
Brandeis University fax (617) 736-2405
Waltham, MA 02254-9110 brenner at auriga.rose.brandeis.eduhttp://www.rose.brandeis.edu/users/brenner