I am purifying a 118 kDa fusion protein (His6-maltose binding protein-my protein). I see induction of a protein at 118 kDa, but also multiple other products are induced that are 75-100 kDa in size. These other products purify with my desired protein, so they must also have a his-tag and maltose-binding protein. I am assuming translation is stopping at various points in the C-terminal region of my fusion protein, but I am already using a codon optimized sequence in Rosetta2 DE3 pLySs cells. Why might I be getting multiple products/How can I fix this problem?
Hi Madison, it is more likely that the lower MW bands are due to proteinases cleaving your protein.
You can see if these bands are diminished by adding proteinase inhibitor cocktail (Roche, Sigma) to your sample after harvesting.
Ok, is there a way to tell which it is? I see these bands even when just taking a sample of my cells (after induction) and running them on an SDS PAGE gel.
I have been using 1mM PMSF, maybe I should add the cocktail in addition?
Thank you for your response.
Eukaryotic protein getting expressed in E. Coli. Cleavage or truncation is occurring at C terminal end; any N terminal truncation would remove tags and protein would not be pulled down on nickel column. Based on my current research these processing events do not occur for this protein.
PMSF is a good inhibitor of serine proteinases, but it needs to be made fresh every time. PMSF stock solutions degrade with time.
Since you are using a his tag. you should try EDTA-free proteinase inhibitor cocktail.
The fusion protein is 118 kda and the other bands are 75-100 kda. All of these products are induced upon addition of IPTG. After cleaving the tag my protein is supposed to be 74 kda and the other bands are around 48-65 kda.
After nickel purification and then cleaving the tag, all the bands remain, and have lost the his/maltose binding protein tag. This is why I was unsure whether the problem was due to proteases or truncated protein products.
I have a antibody for a His tag but not for my protein.
I am not currently purifying from inclusion bodies.
In case anyone is having a similar problem I discovered that the multiple products were caused by truncated protein products being produced due to ribosomal-stalling. My protein has many polyproline stretches (particularly in the C-terminal region of my protein) that are known to cause ribosome stalling. See this paper for more details: Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P.
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