A question I'm finding hard to answer (recombinant protein)

[ibz52002]

Hi guys,

I would be very grateful if you could help me answer the following question. I have an exam coming and i've been trying to do some practice exam questions and I am stuck on this one.

Describe two differences between prokaryotic and eukaryotic transcription / translation that influence a recombinant protein expression experiment when bacteria are used as the host?

Many Thanks for your help,

[JackBean]

do you know what are the respective steps in gene expression in prokaryotes and eukaryotes? There is one step in eukaryotes, which is not used in prokaryotes.

[ibz52002]

No could you please explain?

[ibz52002]

anybody please?

[AstraSequi]

What is the sequence of gene expression that you learned? Can you speculate on what the differences might be?

[JackBean]

Try little bit harder I'm not gonna do your homework, if you're not able even to check Wikipedia

[ibz52002]

Hi guys does this seem right? Can you add anymore to the answer (improve it) ?


A recombinant protein expression experiment involves cloning foreign DNA into a vector for expression of the protein (encoded by the cloned gene) in a host. In this case, the host is a bacterium which is a prokaryote. The main points to be covered in the answer relate to important differences between prokaryotes (the host bacterium) and eukaryotes (a human gene, for example) in transcription and translation.

Concerning transcription:

1 - bacterial promoters are different from eukaryotic promoters. In designing the expression experiment, a bacterial promoter sequence should be selected that is optimum for the desired expression levels and this must be cloned into the expression vector upstream of the foreign gene to be expressed.

2- Eukaryotic genes often contain introns. Bacteria do not have the splicing machinery to splice these introns after transcription. Therefore the sequence cloned into the expression vector must have the intron-coding sequences removed. This is why cDNA is often prepared first.

Concerning translation:

Eukaryotic mRNAs do not contain a Shine-Delgarno sequence which is required in bacteria for correct positioning of the mRNA in the ribosome. Therefore, the DNA cloned into the expression vector must include this sequence.

[AstraSequi]

That looks reasonable.

By the way, the "extra step" Jack was referring to above was probably mRNA or protein processing. Splicing is one example of this, but there are many other types of modifications as well - glycosylation and phosphorylation, for example, which occur after translation. There is no guarantee that the enzymes that do this for the protein in the original species are also present in the expression system, even if the expression system is another eukaryote. For prokaryotes (bacteria), this is even more unlikely since they very rarely have such modifications.

[JackBean]

ibz: yes I was aiming to 2, that's exactly what you have to take into account when cloning eukaryotic gene. The promotor, on the otehr hand, is not of big concern, because it's usually part of your vector, which is specifically constructed for desired host, thus the promotor should work there.

Just to make it clear, the host does not have to be bacteria, it can be yeast, cell culture (either plant, insect or even human) or whole eukaryotic organism.

[adobe]

The RNA Polymerase is different.