membrane protein expression - (Mar/26/2012 )

Hello everyone.. I have been facing a little trouble in expressing two
membrane proteins, alkane monooxygenase (1100 bases) and alcohol
dehydrogenase (1050 bases).. The genes are from Pseudomonas aeruginosa
and I have successfully cloned them both into two vectors: pHIS1 and
pet28 ( and transformed them into BL21 DE3.. The sequencing results of the purified plasmids show that
the genes are present and they are in the correct reading frame. after induction using 1mM IPTG at 0.6 OD for 8 hrs, I am getting a similar protein pattern in both the induced and non induced. I have also tried
varying the IPTG concentration (0.1mM and 0.5mM), incubation
temperature (28 and 30 degrees celcius) and host strain (BL21 DE3
pLysS). Moreover, I have also tried 1% Triton - X treatment, liquid
nitrogen freeze-thawing and sonication of the induced samples. It has
been six months and I have not been successful in expressing both
these proteins. I would really appreciate it if anyone has any
suggestions to solve this..thank you in advance =)

Hi, Can you tell me more details about the resriction sites you have choosen for cloning ? As theoretically there should not be any problem if we r cloning a gene for protein expression in eiother of sites in MCS But practically it affects a lot !!!!!!!!!!

Another thing have u tried C41 or C43 strains ?

Have you checked pellet also ?

Have you done any western blot ? As this will tell us protein truncated or no expression at all ?

Regards

Molecule .