cDNA

[kdawg]

I've hit a snag, im new to my lab and have been assigned a project to investigate te effect of single amino acid changes in a tyrosine kinase. this is my plan of attack
1. extract RNA ffrom tissue and create cDNA
2. PCR amplify gene of interest using primers (do i need to include the start codon in my primer?)
3. insert into an expression vector - overproduction and purification
4 mutagenesis

Thanks

[clevermizo]

Originally Posted by kdawg

I've hit a snag, im new to my lab and have been assigned a project to investigate te effect of single amino acid changes in a tyrosine kinase. this is my plan of attack
1. extract RNA ffrom tissue and create cDNA
2. PCR amplify gene of interest using primers (do i need to include the start codon in my primer?)
3. insert into an expression vector - overproduction and purification
4 mutagenesis

Thanks

Actually, your plan of attack consists mostly of building reagents. Once you have expression constructs containing your gene of interest and/or multiple mutants - what is your assay that will determine the effect of the mutagenesis? Are you looking at its effect on kinase activity? Are you doing a kinase assay? Are you looking at its ability to bind a substrate?

If you are expressing this protein in mammalian cells either your PCR product or the expression vector must contain a 5' Kozak consensus sequence and a start codon in addition to whatever promoter it uses (many mammalian expression vectors use a CMV promoter).

If you are expressing this protein in bacteria, you have to make sure your PCR products (i.e. your primer) includes a Shine-Dalgarno consensus sequence and a start codon.

Furthermore, you should make sure there is a stop codon in your PCR product or in the vector backbone so that you obtain the correct protein.

If you are expressing your protein as a fusion with another protein (like GFP, GSP, 6xHis, Myc, MBP, etc.), your coding sequence must be in frame with the tag, otherwise you will be making something other than what you expect. If the tag is C-terminal, then clearly you should not include a stop codon and if the tag is N-terminal, you should not have a Kozak sequence intervening between the tag and your first codon.

[kdawg]

thanks for that, we're eventually going to do kinase assays, and we'll be expressing in E.coli. Does the shine-Dalgrano sequence need to be upstream of my RE1 site and start codon?
also, when inserting gene of interest into vector with his tag do i need to design new primers for expression that don't include the stop codon?
ugh I'm so confused - been out of the field for a while so its like starting from scratch!

[clevermizo]

Originally Posted by kdawg

thanks for that, we're eventually going to do kinase assays, and we'll be expressing in E.coli. Does the shine-Dalgrano sequence need to be upstream of my RE1 site and start codon?

Well, I suppose you can read about that [Only registered users see links. ].

also, when inserting gene of interest into vector with his tag do i need to design new primers for expression that don't include the stop codon?

Well it depends. Do you not have the expression vector sequence? Is it pET? pRSET?

If the His-tag is part of the vector, is it N'terminus or C'terminus?

If it is N' terminus then obviously the stop codon is ok. Furthermore, if the tag is N'-terminus, then there is a Shine Dalgarno and start ATG ahead of it, so you don't need to worry about adding them to your cDNA. However, you must design primers so that the gene is in frame with the tag. Otherwise you'll have an entirely different protein product than what you expect. The important primer here is the forward primer. The reverse primer doesn't need to make anything in frame with anything else because it's at the end.


For example, suppose my sequence starts out like this:


ATG GAG GGG CGG


And suppose in the vector, the sequence after the His tag has an MCS with EcoRI that looks like this:

CAT CAT CAT CAT CAT CAT GAA TTC

As you can see the frame of the restriction site is just GAA'TTC. So when you design a primer to add the restriction site, it must look like this:

GAA'TTC'ATG'GAG etc.

However, suppose the vector was like this instead:

CAT CAT CAT CAT CAT CAT GGA ATT CGG

If I just clone in my sequence as is it will be out of frame:

CAT CAT CAT CAT CAT CAT GGA ATT CAT GGA GGG GCG G

In order to keep it in frame with the tag, I will have to make a primer that perhaps adds a nucleotide or two to maintain the frame:

GA ATT C__ ATG GAG GGG CGG

In this case, as you can see I need 2 nucleotides to maintain the frame. They could be the same ones from the vector backbone I suppose.

GA AAT CGG ATG GAG GGG CGG etc.

Now, when I perform the ligation, this will be in the appropriate reading frame. I should obviously sequence my final construct to make sure that the gene of interest is in frame with the His tag.


On the other hand, if the Hist tag C'terminus then the stop codon would interrupt your protein and the tag, so you'd need to remove it by designing new primers. Furthermore, you would need to make sure you have a Shine-Dalgarno and start ATG ahead of your gene. And finally, your reverse primer must maintain the reading frame as above with the C' terminal tag.