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
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