Hi everyone
we tried to express a fusion constract (4 attached gene) in pet21a plasmis and E. coli.
But we couldn't see any 75 kda band in SDS-page.
we tested any optimization like different OD, IPTG and etc.
sequence and ORF are OK
do you have any suggestion?
Manuele Martinelli
Dear Shaho
expression of 4 fused ORF could be challenging since is it possible that during the folding process the protein is not well folded and/or linkers may be esposed to proteases activity.
1) Which linker did you use?
2) Can you share a picture of the gel to see if the are some bands corresponding to the degraded proteins?
3) Did you tried a single gene order or more combinations?
4) Do you know if the single genes could be expressed in E.coli?
best regards
Manuele
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