today in the GGS LIVE section we will learn how to virtually design and generate a fusion protein.
Method: protein tagging.
About: protein tagging allows to perform specific experimenents that are not possible with the endogenous protein (wild-type protein).
What: desing and generation of protein X fusion with a GFP (green fluorescent protein) tag.
There are many different tags available for protein tagging. Choosing the tag depends on the experiments that we want to perfrorm with the fusion protein. So, if we want to:
- purify the protein of interest, we would use MBP (maltose binding protein), GST (gluthatione S-transferase), FLAG or His (hexahisitidine) tags,
- easily detect our protein, we would use epitope tags like myc, V5 or HA,
- follow cellular localization of the protein, we would use fluorescent tag like GFP.
The most important is to remember that fusion protein might behave differently in vivo than wild-type protein (folidng, solubility or activity, etc may change) so it is crucial to check if the fusion is functional before we start our experiments.
Ok lets start with design of the fusion protein. In our study case we will use a sequence of a protein X (shown below) and a pEGFP-C1 and -N1 plasmids for N and C terminal tagging, respectively.
As you see there are many restriction sites available in both plasmids. Our aim here is to find a restriction enzymes that will cut in MCS of our vectors but not in the sequence of protein X (we can do that with any cloning software, like free pDRAW32 which you can download from here). In our study case two enzymes XhoI and EcoRI are cuttining in the MCSs but not in sequence of protein X. What we have to do now is to virtually introduce XhoI and EcoRI sequences at 5' and 3' end, respectively (sequences recognised by XhoI and EcoRI endonucleases are available here XhoI and EcoRI). There is one more issue to look at before we are going to add our restriction sites (see picture below).
It is important to remove start codon of the protein when tagging it on the N-terminus to avoid an expression of untagged form of the protein. You have to remember that promoter will drive expression of any open reading frame that is downstream of it. It is also crucial to remove stop codon when we tagging protein on the C-terminus to prevent premature termination and allow expression of a fusion protein. Including above information we have:
Now we put this sequence into our plasmid and we get this:
Lets have a look at our constructs now. We are going to focus on the reading frame at the moment. In the case of N-terminal tagging our reading frame is determined by the two last codons of GFP tag. To get the right fusion DNA sequence of protein X has to be in the reading frame with the GFP tag. The reading frame is indicated by the horizontal brackets (each triplet codes for one amino acid). As you can see stop codon of the protein X (indicated with the red colour) is not in reading frame with GFP tag. To shift the reading frame we have to add two extra nuclotides to our protein X just between the XhoI restriction site and the coding sequence of protein X. Similar situation takes place with the C-terminal tagging. You can see that now start codon is not in frame with GFP tag and addition of a single nuclotide between EcoRI restriction site and protein sequence will rescue that problem (see the pictures below).
It is important to remember that addition of extra nuclotides to our sequence may result in introduction of the stop codon. We have to check our sequence before we proceed further. If everything is ready we should obtain this:
As you can see now, protein X sequence is in the frame with the GFP tag in both cases. Now the protein sequence including the XhoI / EcoRI restriction sites and extra nucleotides can be used to design primers for cloning of the protein X DNA. Such DNA then will be sequenced to check for potential mutations and if correct subcloned into pEGFPN1 or -C1 plasmids. Such construct can be later used for expression and localization studies of the protein X.
I hope u enjoyed it.
Maciek
GGS TEAM
I'm a total beginner in this field, and I can't tell you how much this helped, thank you so much!
ReplyDeletenot a beginner in this field but am embarrassingly lacking in my skills as a true molecular biologist. this is very well-written and helpful!! thanks!!
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