I have a protein that is 117 amino acids long, with a 21 amino acid transmembrane domain at the C-terminus. I've cloned this protein into the pET28 expression vector in BL21, but am not seeing any protein expression. Do you think the transmembrane domain could be the reason for the lack of expression? What strategies can I try to improve the expression of this transmembrane protein?
## Background
- Protein length: 117 amino acids (from virus)
- Transmembrane domain : C-terminus, length: 21 amino acids
- Expression vector used: pET28
host:BL21
- Current expression status: No detectable protein expression
Dear Nada Mohammed Saleh
present of transmembrane domain can certanilly reduce a lot the expression of the protein in E.coli (but even if other more complex organisms) seems the transmembrane space is limited and the overexpression of a protein in the membrane can result toxic to the cells.
the simple solution is try to produce the soluble portion of the protein by excluding the C-terminal transmembrane part and also the N-terminal signal peptide, if present.
However there are other possible factors that can make difficult the expression of the soluble domain in E.coli that are:
- Presence of rare codons in the DNA sequence codify for the viral protein (that could be overcome using a E.coli strain as Rosetta, codifying for rare codon, or using an optimized codon DNA fragment as template for cloning
- Presence of many cysteines involved in S-S bonds. Since the E.coli cytoplasm is not able to form S-S bonds and the protein can be expressed as insoluble in IB, if unfolded.
good luck
Manuele
Dear Nada Mohammed Saleh
Hello, I agree with Manuele Martinelli's opinion. Most membrane proteins have pretty low expression in E. coli. When I purified membrane proteins in E. coli, I used the inclusion body fraction and membrane fraction to purify my protein. After purifying the fractions using Ni-NTA, I finally saw my protein's thin band on the SDS-PAGE gel. You might not see your protein band in the supernatant after cell lysis. But, you might see your protein band after purification if using the inclusion body fraction and membrane fraction.
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