Heterologous expression of a bacterial gene in BL21 cell is generating two protein of different size: one of full length and another one of smaller size. The possibility of fragmentation post-translation has been ruled out as the immunoblotting of lysate using anti-sera generated against full length protein gives detection of only full length and smaller size protein (no detection of larger fragment). Kindly give your suggestions: (1) is it due to leaky scanning which has been well reported in eukaryotes and viruses? (2) or it is something else?
This sounds like leaky scanning to me. I would check your ribosomal binding sites to make sure that your sequence is fully optimized to translate the entire protein. I have witnessed this first hand with large proteins I have tried to express in BL21.
Visit the following website and create a free account https://www.denovodna.com/software/predict_rbs_calculator. Type in your entire RNA sequence and you will be provided with a series of possible ribosomal binding sites and the rates at which each one will translate at. You can actually compare which ones are better than others and back calculate the size of the protein you would get from different RBS's to see if the smaller sized protein you are getting is due to leaky scanning.
Best of luck,
Max
Hello Saddam,
l disagree with your interpretation ruling out post-translational cleavage of the recombinant protein. Failure to detect the missing larger fragment could be due to the fact that it lacks epitomes reacting with the antiserum. Alternatively, it could also be missing because it is destroyed in vivo by E. coli proteases. But checking the possibility of alternative translation start sites, as suggested by Max, is also a good idea. Such sites would have to be in the proper reading frame, though.
Pierre Béguin and Max Levine thank you for your kind suggestions. It is very unlikely that a polyclonal antibody raised against full length protein will not detect the fragments of the same protein. However I have already checked the all possible antigenic epitopes in full length protein using online servers and it is almost equally distributed throughout the protein. Is it possible that an E.coli protease specifically degrading the larger fragment but not the full length and smaller fragment?
Searching for all the possible and strong start codon is a good idea, in fact I have done some work in this regard also, manually, but with reference to criteria of a strong start codon in eukaryotes and results are not matching with the size of my smaller protein. But I will do the suggested one again.
Further inputs will be very helpful
Thanks a lot
If you want to have a definitive explanation, try to get sequence data, either by Edman sequencing or by mass spectrometry of a tryptic digest of the two species that are produced by your recombinant clone.
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