I intend to express a protein (enzyme) from a bacteria after PCR amplification and transforming into E.coli. The target gene is from a cold adapted bacterium and the enzyme is active at 10 degrees.
1. What should be my consideration during PCR amplification of the target gene. ( I dont want to create cDNA library and screen for my target CDS). Should my PCR product contain only the coding sequence?
2. What about the UTR's? Should I consider the 5' and 3' UTRs while designing primers for the target gene. Should my PCR product span the (5'-UTR-coding sequence-3'UTR) region or just the coding sequence starting from start codon to stop codon. (Although an expression vector contains RBS and strong promoter)
3. Will the upstream and downstream forward and reverse primers of approx ~ 18-20 nucleotides containing restriction sites put for manipulation alter the reading frame of my target gene or it does not matter. Will the protein get expressed or folded properly?
4. E.coli which generally needs 37 degree (say BL21 (DE3)) for growth. what about my protein which is active at 10 degree, what will be its fate?
I am hoping to get clarified on these points, I may not be that expressive regarding my problem, but I believe you can figure out my dilemma.
Thanks everyone!!!
1- Yes, only the coding sequence.
2- Depends on your purpose. Apparently you want to express this enzyme in E. coli so no, you only need the coding sequence.
3-re: RE sites on primers. Yes, the most straightforward approach is to include them so as to be able to clone directly into an expression vector. 18-20 bp is a little tight for that, by the way; you can make longer primers You must also leave extra nucleotides 5' to the RE site (most RE cannot cut if their recognition site is flush at the end of a ds-DNA fragment). re: Will the enzyme fold properly? Heaven knows...seriously, it's difficult to know beforehand. If your enzyme relies on disulphide bonding then no, it will not fold. If it relies on a folding partner that is absent in E. coli then no, it will not fold. If it's part of a complex and is never found in monomeric form then no, it will most likely not fold properly. If it has lots of proline residues in cis conformations then it will most likely. not fold properly. If it wraps around a cofactor involving lots of hydrogen bonding and/or salt bridges and that cofactor is not there in E. coli no, it will not fold. If it folds slowly and exposes long-lived hydrophobic patches then no, as soon as it is expressed at high levels it will aggregate rather than fold properly. And so on...
4- Same as above, you'll have to try. BTW, TaKaRa sells expression vectors with the cspA promoter (induction at cold temperatures), they might be worth a try.
hope it helps
Hi Rajal
Here are a few pointers:
1. Before you start, do a codon optimization. If your gene contains a number of rare E. coli codons, this can significantly reduce the level of expression you can obtain.
2. Clone your gene in a commercial vector like pET where you can choose all kinds of features that are suited for you specific requirements (i.e. a good ribosomal binding site, inducible promoter etc)
3. Design primers so they do not change the coding sequence. Introduce whatever restriction sites your vector requires.
4) Difficult to say about enzyme activity. You can grow E. coli at 10C but it will be very slowly and the yield will be low. However, I would just try 37C or 30C and see what happens.
Good luck
Jesper
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