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Bacterial expression vector for secreted proteins

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Jan 17 03:44:53 EST 2000


Student wrote:

> Can anyone recommend a good expression vector that can help me produce lots
> of proteins in the culture supernatant? Will it work if I put a, signal
> peptide (e.g. pelB), in front of my protein and ligate it to one like pET?
> Or is a special vector required for secreted proteins?
>
> many thanks.

Hi,
We are using the pEZZ18 vector (pharmacia) for production of secreted protein
in E.coli. It carries the signal peptide form S.aureus proteinA and a ZZ-tag
(ie. two IgG-binding domains of proteinA, great for purification), and allows
for the synthesis and secretion of, in our hands, proteins up to 40kDa (ie.
55kDa in total with the 15kDa tag). We never succeeded in making bigger
proteins, but that does not necessarily say it is impossible. I wouldn´t try
it, however, if your protein is >70kDa.
As to the yield, it depends on the protein and the codon usage of the coding
DNA. In our hands, we can get milligram quantities in normal culture
conditions (like 5mg from 1l of LB), and lots more by fermentation (200-300mg
per 10l).

hope this helps,
Frank





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