Hello everyone, I am trying to find an efective method to express the glycoprotein spike (S) or partial S (S1) of PED virus. My lab is full equipment and used to express by E.coli system. However, I cannot find any paper, juournal or document for this issue. They do express on baculovirus (not much yeild), mamamilan cells (a little bit higher). But nothing related to E.coli sys tem. And I wonder why? Is there anyone have experience in this kind of expresion?
Dear Thu
i'm not an expert of PED virus and on its specific glycoprotein however as a general comment, i think is not impossibile express this protein in e.coli (attached you can find a paper where they express the Spike S from one other virus) but the problem is that the e.coli is not able to perform the glycosilation and are you sure that the non glycosilated protein can work as the glycosilated for your esperiments? Because it seems that its function is related to the glycosation. i'm right?
i'm actually working with expi293 cells, which are expensives but amazing in terms of growth and recombinant expression. For some luxky proteins i obtained yields from 0.2 to 0.5 mg to ml of culture which is not amazing only for mammalian cells but also difficult to reach with e.coli. Of course with those cells, You need to work more clean than e.coli to avoid bacterial contaminations and you need a shaker with CO2 but for the rest, expi293 growth in shake flask up to high od, are simple to be trasfected and able to secrete the protein into the media that make purification very simple.
in some way are more similar to e.coli than from the most of mammalian used until sone years ago for recombinant expression.
i know that if your lab is focus on e.coli but i'm sure that the intricmduction of this system will represent a huge step up not only for your project but for your entire group.
good luck
ciao.
Manuele
Hi. Thu Hien Thi Nguyen.
What kinds of subsequent experiments or researches do you want carry out with your spike protein? In my lab, E.coli system usually is used for producing protein which would be purified by the his-tag or Mbp-tag. You know that, recombinant protein can be expressed in many systems. E.coli expression system belongs to prokaryotic expression system. There are many disadvanteages in prokaryotic expression system compared with other systems, such as non-functional multi-domain eukaryotic protein.
You can google the detailed comparison amony these expression systems. (Thermofisher offer an excellent overview, you can check it out)
The eukaryotic expression system can produce the more natural-like protein. It is more efficient and common during the transfection in cell lines for further study.
Good luck and enjoy your researches.
As mentioned in the previous comments, the E. coli expression system is not conducive to maintaining the activity and natural conformation of eukaryotic-derived proteins. If you have the conditions, try to use eukaryotic expression systems, although the construction of the expression system is a bit more complicated, but it is helpful for subsequent experiments. If you don’t have enough conditions, you can consult Creative Biostructure, which I know is a very professional supplier of protein biotechnology.
Good luck!
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