Hello Christopher!
Currently I am working on a similar cloning challenge - and also 293 or 293T cells, dependent on the selection marker of the vector. The leader peptide sequence of interleukin-2 (IL-2) is one possibility and used in the lab in which I am working.
Good luck with your experiments!
Hi Christopher,
There are several signal peptide sequences that can be used for this purpose (sometimes the leader sequence of your protein is the one that works better!!). However, the most used in our lab is the signal peptide sequence of CD33: MPLLLLLPLLWAGALA because it is quite short and effective.
I hope this information is useful for you.
Good luck
Jorge
Thanks for the useful information from all of you. Dr. Henke, is this the IL-2 leader sequence you use?
MYRMQLLSCIALSLALVTNS?
You can also pull up the polylinker of the plasmid pSecTag2 from Invitrogen. It has an IgG kappa light chain leader sequence that works well. I use a mutated form of this sequence (contains the AgeI site at the 3' end) and cloned it upstream of my protein in pcDNA3.1+Zeo and had success with that vector as well.
Hi Christopher
I have used IL-2ss for recombinant protein expression in baculovirus system. It also worked very well for transient protein expression in 293T cells. Good luck in your experiment!
Hello!
After some searching I found that we also use the vector pSecTag2 from Life Technologies that contains a murine leader sequence based on the Ig kappa chain.
Sequence should be 21 aa:
MAQVKLQESGTELAKPGAAVK
I was told that a previous student used this with great results.
Hope this helps!
Hi everyone.
You can take a look at these two papers:Improving mammalian cell factories: The selection of signal peptide has a major impact on recombinant protein synthesis
and secretion in mammalian cells
and
Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression
We have used both for recombinant protein production with good yields.
My personal experience is that the Gaussia SP works exceptionally well in most cases.
Gernot
Hello!
Is AA seq of certain signal peptide under IP protection. How can I find it out, is there any database or smth?
If the peptide sequence is occurring in nature, it should be difficult to protect it.
I know this post has been here since 2013, but I recently found a platform by DTU (called SignalP) very helpful which I would like to share for the sake of interested learners.
I've come to experience that the efficiency of a signal peptide depends on the nature of the GOI. This is why the SignalP tool comes in handy. Just put in the full amino acid sequence, including the leader sequence, and it will inform you of the secretion efficiency. You can keep changing the SP until you get a score >0.8. Check it out!
http://www.cbs.dtu.dk/services/SignalP/
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