trouble with membrane protein expression

heraa rajandas via methods%40net.bio.net (by heraadaas from gmail.com)
Mon Mar 26 02:56:12 EST 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 (b).. The sequencing results of the purified plasmids show that
the genes are in frame (ie. the reading frame is correct and no stop
codons are present within the gene). Initially, I carried out the
induction using 1mM IPTG at 37 degrees celcius. 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 =)


More information about the Methods mailing list