Help! Protein Expression in E. coli
MAD2631 at rsgis6.tamu.edu
Mon Mar 11 16:20:04 EST 1996
I am trying to express two cDNA clones we isolated, in E. coli BL21 DE3
to make antibodies. Both these cDNA clones (proposed to) code for highly
glycosylated proteins in plants (both have N terminal signal sequence
which has been deleted by PCR amplification of the rest of the cDNA and
cloning in pET30 expression vector) and have a hydrophobic C terminal.
I seem to get very low level induction (as per S tag western) but the
molecular weight of the product is about 10 kd higher than expected (The
expression vector with the cloned gene cannot have a longer reading
frame because of stop codons in other reading frames). I need help in
two areas. 1. Does any of you have any suggestions to increase yield. 2.
Is it possible for proteins to run at higher than expected molecular
weight level in SDS-PAGE? I am new to protein chemistry and would
appreciate all the help I can get.
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