[Nescafe]

Hi,

So I read this:

" human cDNA was c-terminally fused to the peptide fragment using a commercially available cloning vector that included a neomycin selectable marker"

No idea what this means? Can't one use native chemical ligation to fuse a peptide to the c-term?

Thanks in advance,

Nescafé.

[Arkcon]

Well, there's a lot happening in your little text snippet:

" human cDNA

a complimentary DNA sequence isn't a peptide.

was c-terminally fused to the peptide fragment using a commercially available cloning vector

you can make the cloning vector, but you can save time by using a commercially available one

that included a neomycin selectable marker"

when you sub-clone the DNA of interest, with a vector DNA sequence, you want to know that you've succeeded in getting it into a living thing for expression.  The vector usually encodes some "marker" that make success obvious in cell culture.  But that's all separate from your question:

Can't one use native chemical ligation to fuse a peptide to the c-term?

I don't recall of any native method of ligating a DNA sequence to a polypeptide.  I'm sure it happens, but I don't know where.  You don't mention how its done in this case anyway.  But this is a little tricky.

[Yggdrasil]

Do you have a link to the context for that sentence?

Essentially, what I think is going on is that the cDNA for the gene is being inserted into a plasmid.  This plasmid has been designed so that it will produce an mRNA that produces a protein that contains the peptide at the N-terminus and the protein encoded by the cDNA at the C-terminus.  This process (splicing DNA together) is the typical means of producing such "fusion proteins."  Very rarely are fusion proteins synthesized by ligating two proteins together.

[Nescafe]

Do you have a link to the context for that sentence?

Essentially, what I think is going on is that the cDNA for the gene is being inserted into a plasmid.  This plasmid has been designed so that it will produce an mRNA that produces a protein that contains the peptide at the N-terminus and the protein encoded by the cDNA at the C-terminus.  This process (splicing DNA together) is the typical means of producing such "fusion proteins."  Very rarely are fusion proteins synthesized by ligating two proteins together.

You are correct it is a fusion protein.

"Human SSTR2 cDNA (GenBank AY236542) was C-terminally fused to the ProLink™ peptide fragment of β-galactosidase using a commercially available cloning vector that includes a neomycin selectable marker (DiscoveRx). This fusion protein was then subcloned into an MMLV retroviral vector carrying a neomycin selection marker, and the CHO arrestin parental cell line was infected with virus using standard techniques. Clonal cell lines expressing both the SSTR2 GPCR and the human β-arrestin2 were generated using double antibiotic selection and limiting dilution cloning. Clonal cell lines were analyzed for functional responses to agonist and passage stability out to 20 passages."

Here is the full article http://jbx.sagepub.com/content/13/8/737.long


I am still a bit confused, this is a bit molecular biology heavy for me :|

Nescafe.

[Nescafe]

Do you have a link to the context for that sentence?

Essentially, what I think is going on is that the cDNA for the gene is being inserted into a plasmid.  This plasmid has been designed so that it will produce an mRNA that produces a protein that contains the peptide at the N-terminus and the protein encoded by the cDNA at the C-terminus.  This process (splicing DNA together) is the typical means of producing such "fusion proteins."  Very rarely are fusion proteins synthesized by ligating two proteins together.

So reading your post and Arckon's again I think I have a better understanding now. So the cDNA has the genetic sequence to encode the protein in prokaryotes (by the way why do they always express in prokaryotes and not eukaryotes?). They fused the C-terminal of the cDNA with the prolinker. I also understand the marker idea based on what Arckon said, but how do they go about fusing the C-terminal of one cDNA with a peptide? is it a chemical reaction of some sort what is that technique called?

Nescafe.

[Yggdrasil]

The cDNA is not fused to the peptide.  The cDNA is fused to the DNA encoding the peptide.  This is done through standard use of restriction enzymes and DNA ligase.

As for why bacteria are used for expressing proteins, it is much cheaper to grow large amounts of bacteria than eukaryotic cells.  Some proteins (especially transmembrane proteins), however, do not express well in bacterial cells.  This is likely the case with the SSTR2 protein studied in the paper, which is why they use Chinese hamster ovary (CHO) cells for expression rather than bacteria.

[fledarmus]

There are also some post-translational processes that are specific to different cell types, like methylations or binding to sugars. The protein that you get from a bacterial cell is not always exactly the same as the protein you get from a mammalian cell like a CHO cell, even if the codons are the same.