Yield with pTrcHis vectors

Mon Jul 12 07:33:00 EST 1993

Can anyone help with optimising expression of recombinant
proteins with Invitrogen's pTrcHis vectors? We are trying
to get milligram quantities of a plant dioxygenase using
these vectors for expression of His-tagged fusions in E.coli.
So far, we are getting pitifully little active enzyme. We
know that the enzyme is capable of folding in Ecoli, as we've
had expression from a B-galactosidase fusion in gt11. We
don't see a huge quantity of unassembled poylpeptide as inclusion
bodies - the problem seems to be transcription, translation
or stability of the protein. 

We've improved expression a few tens of fold so far by growing
the cells at 25-30 C in 2xYT medium plus sorbitol/betaine. Are 
there any other tricks we can try to bump up expression? As a side
note, the expression of the positive control supplied by Invitrogen
isn't stunning, as least not compared with fusion proteins
I've produced using pUC18 (up to 30% of total cell protein).
Another oddity is that we get some activity from our negative control,
a construct in (supposedly) the wrong reading frame. Ever seen this?
Finally, can adding cofactors help enzymes fold in Ecoli? Our enzyme
requires an Fe ion for activity.

Thanks for any help.

Long Ashton Research Station, Bristol, UK

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