Hi All,

I am looking for some vectors wih different secretion signals to express a protein in HEK293 cells. We have a vector containing the IgG secretion signal but it does not seem to work for a particular protein. We would like to cross-check expression with different signal peptides. Is anybody willing to send me some vectors?

Thanks!

Patrick

Dear Patrick,

we have pTT21 from Yves Durocher with VEGF signal. We signed an MTA, so I can't really send it out. Just to let you know about VEGF.

What about pHLsec from Oxford? This plasmid is freely available. It contains this secretion sequence: MGILPSPGMPALLSLVSLLSVLLMGCVAE, with just the final E being left after cleavage during secretion. I do not know exactly the origins of this sequence, but the secretion works well with it. Besides pHLsec, I also use pTT plasmids from Yves Durocher, also on MTA, so these are not available freely, but the secretion sequences are nothing secret, I believe, and the corresponding DNA could be synthesized, if need be. Secreted alkaline phosphatase (SEAP) is a model protein for pTT plasmids, it uses its own secretion signal, which is for sure not secret.

For secretion, we often use the pSecTag vector from Invitrogen which contains the kappa light chain signal peptide. It works well, but I'm afraid I can't send you the vector...

Thank you Malin, Ondrej and Sabine for the suggestions; I'll have a look at the vectors. We already have a few of the pTT vectors from Durocher, but not the pTT21 plasmid.

Patrick

Hi Patrick, 

The µ-phosphatase signal peptide from pHLSec can also be found in pOPING (pTriEx2 backbone) that can be used for In-Fusion or SLIC. This peptide has, in the few cases that we have tried here in IRB, out-performed the native leader for each protein. It was also benchmarked vs. pHLSec in Oxford. and from memory performed at least as well in HEK.

pOPING is from OPPF/Oxford Uni under MTA the same as pHLsec.

Good Luck

Nick

 

pOPING and a large number of the other OPPF vectors can be acquired through Addgene for a small admin fee (www.addgene.org).

 

Jo

Hi Nick and Jo,

Have any of you tried pOPING for secretory proteins using the baculovirus/insect cell system? We seems to have trouble secreting proteins with this signal peptide in insect cells. Appreciate your thoughts on this.

Thanks,

Linda

Hi Linda,

Many years ago we did produce a secreted protein using pOPING in insect cells, however we are generally using it for HEKs.  This is because large scale transients are easy to do with the HEK system and we can modify the glycosylation using kifunensine or HEK 293S for structural studies.

If the signal sequence isn't working, you could use the native signal sequence and clone into a vector with a C-terminal His-tag such as pOPINE.

Hope this helps,

Jo

Hi Linda, for insect cells we usually add Bip or honeybee mellitin leaders, which tends to work well. But I agree with Jo, better to use HEK for secreted proteins. Succes rate, yields and quality of the secreted proteins are often markedly better in HEK... Best, Rene