How are you targeting the protein to the membrane and to which one? Which strain of E coli are you using? BL21(De3) may not be the best. Especially if the gene is not codon optimized. You could try Rosetta cells. Furthermore, you may get better expression with the Walker Strains (C41 and C43) which were developed for membrane proteins.
Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 http://www.sundberglab.org/Home.html -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohamed Noor Sent: Wednesday, July 13, 2016 4:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic: Expression of human single-pass membrane protein Dear all I am working on a single-pass MP (about 20 kDa) from human that is located in the mitochondrial membrane. I have been expressing it in E. coli but the yield seems low. Since the His-tag is at the N-terminus, there is also degradation on Western blot that very likely corresponds to truncation products (premature release from the mRNA??). Expression was induced with IPTG at 25 C in LB medium. Between switching the expression to a different host and doing codon optimization experiments for expression in E. coli, what is your overall experience in this situation? The TM region is essential for function, so I cannot express just the soluble portion for activity assays. Thanks. Mohamed
