For my project, I am attempting to purify an intrinsically disordered domain cloned in a pET28b vector. The strain used for overexpression is Rosetta II(DE3).
Firstly, I encountered an unexpected size difference on SDS-Page. The theoretical size is 17606.27 Da, but the observed size was around 28 kDa. I suspect this variance may be attributed to the highly charged sequence.
Secondly, I performed purification using Ni-NTA and SPFF columns. The protein was verified by LC-MS, revealing a mass of 25,662 Da, which is unusual. The DNA sequence is correctly inserted in pET28b, however, the T7 terminator is located relatively close to the stop codon (12 nucleotides away).
Consequently, I am unsure if this proximity could affect the transcription/translation of my protein.
Another possibility is a frameshift problem within the sequence.
What other explanations could account for this phenomenon?
Read-through of the stop codon is a possibility. Calculate the mass if the intended stop codon was read through and the next stop codon was reached.
Also consider whether the wrong protein was cloned or purified. Re-sequence the plasmid insert to make sure it is correct. If so, try to get a bit of internal amino acid sequencing done to see if it matches the intended protein.
I have been hit by this before. I suggest sequencing your plasmid and look for large primer insertions. It is totally possible to have correctly designed primers for molecular cloning and yet still get massive in-frame primer insertions into your protein which have no affect on the expression or purification or folding or activity of your protein but increase the number of amino acids in your protein and thereby increase the mass of your protein.
If the terminator interfered with proper translation termination, you would get an additional 4 amino acids from that terminator sequence, plus another 10 added via trans-translation from tmRNA. However, this doesn't in itself explain your size discrepancy. What does the 5' end of your gene/insert look like? Perhaps translation is starting from another start codon further upstream of what you expected. Also, do you glean any sequence information from your LC-MS? How sure are you that it's your protein, and not a contaminant?
Thank you for all this valuable information.
In response to Devin Camenares , from the end of my T7 promoter to the first start codon, there are only 68bp. I've checked as you suggested, but no other start codon is localized in that region. Although I didn't obtain the sequence from the LC-MS results, the sample underwent a two-step purification process (Ni-NTA followed by SPFF), and verification was performed via SDS-page. Therefore, I believe it's my protein and not a contaminant.
For some supplementary information, my plasmid was ordered. I've checked the frame and sequence, and it seems correct. I will test the protein production with another strain of BL21 to see if the problem could arise from my strain background
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