mutant protein expression & purification problem in E.coli - Protein with five residues mutated to Alanine's (Apr/19/2007 )
our lab has been using E.coli (BL 21 pLysS strain) to express a viral protein wild-type without any problem. however, after I mutated five amino acids (residues all mutated to Alanine's) in the entire protein, I have problem with the protein expression and purification in E.coli now.
The expression seems to be very low and the yields of purification with the Nickel column (FPLC) is much much lower compared to the wild-type protein purification.
Our viral protein is his-tagged at the C-terminus (as the His tag at the N-terminus yields inactive protein). The expression plasmid (pET vector) is Amp & Chlor resistant and doesn't require induction.
I also checked the mutated residues in my viral protein, the mutated residues don't have rare codons for E.coli. I am wondering what causes my mutant protein expression & purification problem. If anyone has the similar experience, would you share it with me? any advice/suggestion is appreciated!
ps: the size of our viral protein is about 40 kDa (His tag included)
Tony
Our viral protein is his-tagged at the C-terminus (as the His tag at the N-terminus yields inactive protein). The expression plasmid (pET vector) is Amp & Chlor resistant and doesn't require induction.
Sorry, but what do you mean you don't require induction? If you don't add IPTG, you certainly won't get much protein.
There are some kits that allow production of protein (such as GST fusion proteins) without induction by IPTG. The protein expression will be induced by bacteria density. Have to use BL21 though. Maybe that is what he mean by without induction?
There are some kits that allow production of protein (such as GST fusion proteins) without induction by IPTG. The protein expression will be induced by bacteria density. Have to use BL21 though. Maybe that is what he mean by without induction?
sorry about the confusion. Yes, the viral protein expression plasmid doesn't need induction. It expresses wild-type protein very well at 37 celsius overnight. I just have problem with expressing the mutant viral protein.
Is there anything unusual about the codon usage in the mutated protein? Have you used the same codon all the way through with the substitutions? Are the Ala mutations close to each other, or well-separated? You might find that you are exhausting the local tRNA population if you are using the less-common codons (GCU or GCU), especially if you have a run of Ala residues. How is the expression controlled?
The expression of the fusion protein in this case is due to density. With low OD, the gene is not activated. When there are more cells (log phase), gene with be activated and express fusion protein. This way you don't have to induce and it take only one day grow and then purify protein instead of 1 day grow and 1 day induction as normal.
As for the low expression of the mutant: since you have a run of Ala, maybe the protein itself is unstable? It might be degraded by the bacteria. Try for shorter time or maybe come back to the normal type of IPTG induction to see how?
Bad answer, BL21pLysS strain contain tRNA for rare codons....