pET expression problems

Adrian Clarke adrian.clarke at plantphys.umu.se
Thu Sep 3 07:32:46 EST 1998


I have a question that I hope someone can answer.

I have used pET23a as cloning vector for over expressing an Arabidopsis
thaliana protein. I have 4 different constructs: One where the resulting

protein is predicted to be 33 kDa + 6 C-terminal histidines (a native
N-terminal); another where the N-terminal has a T7-tag (12 amino acids I

think); and two constructs the same as the above, but with shorter
N-terminals (corresponding to 30 kDa Arabidopsis protein). Control
sequencing of the constructs was performed to ensure the right
translation. These recombinant proteins are HIGHLY toxic to E.coli,
especially the
full length clones. Trying to transform BL21(DE3) with these constructs
resulted in no transformants! (but a control with the vector was fine).
I then tried a "super safe" system with BL21 first transformed with a
plasmid containing the T7 polymerase behind a temperature regulated
promoter (induction at 42°), then my constructs.
With this system I obtained over-expressed protein as inclusion bodies
(around 1.8 mg/1 litre of culture).

The problem I have is that the sizes are strange - around 25-26 kDa for
all constructs when I run a SDS-PAGE gel, whereas I expected 33-34 kDa
from
the full length clones and 30-31 kDa from the others. In the protein gel

system I use it is possible to distinguish a 35 kDa polypeptide from a
30 kDa
polypeptide. Amino acid sequencing of one of the over-expressed proteins

showed that it is the right protein! Running Leaf extracts on a Western
gel and probing with antibodies against this protein results in a
polypeptide band of approximately 34 kDa.

Right now I have no idea what is happening. Does anyone else have
experience with these expression systems and the pET vector? Is it
possible that E.coli is processing the over-expressed protein? Any
helpful advice or ideas would be greatly appreciated.

Adrian

--

Dr. Adrian K. Clarke
Associate Professor
Department of Plant Physiology
University of Umeå
Umeå S-901 87
Sweden
Tel: +46 90 7865209
Fax: +46 90 7866676





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