You are interested in a eukaryotic protein involved in immunity, and you are attempting to express this protein in E. coli in order to produce large amounts of the protein

Question

You have identified the gene and place a copy of the gene on a plasmid in E. coli next to a bacterial promoter sequence. You determine that lots of mRNA is made from your gene in your E. coli system, but the protein produced is larger and doesn't have the same properties as the eukaryotic protein you expected. What mistake have you made and how can you fix it?
What will be an ideal response?

Answer 1

Answer: Eukaryotic mRNA goes through extensive processing compared to prokaryotic mRNAs, including the removal of introns after transcription. Transcription appears to be working in the E. coli system, but the introns are still present in the mRNA when translation begins because prokaryotic translation begins as soon as the mRNA is produced. In order to fix the problem, you would need to determine the final sequence of the mature mRNA for the protein (after all introns are removed) and insert the DNA sequence without the introns next to the bacterial promoter on the plasmid. Other sequences, such as a 3' polyA tail or a 5' cap should not be necessary in a prokaryotic system.