As we discussed at the workshop in Barca (thanks to Nicholas et al for a great meeting!), we are interesting in trying out Takara's pCold I-IV and pCold TF expression vectors.
Paper: Qing G et al, 2004, Nature Biotechnology.
Brief summary of principle: Vectors harbour the cspA promoter, thus, at log phase expression cultures are equilibrated to 15 degrees and induced with IPTG and continues shaking for 24 hours. Vectors encode unique features, such as Protein S tag, his-tag, translation effiency element and/or Trigger Factor (TF).
What is your experience with the pCold system?
Tags may be removed by Factor Xa, Thrombin or HRV 3C Protease. What we would like for our pipeline is a TEV-cleavable tag. I remember we discussed making a TEV-cleavable version of the pCold TF was troublesome but that a increased linker solved the problem? How was the linker?
There is also a GST-tagged version of pCold I avaiable, although not commercial: Hayashi K et al, 2008, PEP.
We have used the pCold-TF (Trigger Factor) vector - actually modified versions of it containing our LIC sequence. We have made two versions; (1) where we removed the factorXa and thrombin sequences and instead use a 3C protease sequence (his-TF-3C-LIC) and (2) where we added a strepII-TEV site before the 3C site (his-TF-TEV-strepII-3C-LIC) to increase the spacer between TF and our protein. Here's a small summary:
- Expression at 20 °C or lower gives you a large amount of protein (20 to ~ 100 mg/l).
- Sometimes the protein expresed from (1) could not be cleaved by 3C protease; therefore we came up with the enhanced linker (2). Athough this improved cleavage in some cases, in others it still did not cleave the protein. May be it is a signature of (partially) unfolded protein.
- In one example where expression was difficult due to degradation, expression in the pCold vector significantly reduced degradation.
- In at least 2 cases proteins could only be expressed in the soluble fraction when the pCold vector was used (meaning that protein remained soluble after cleaving the his-TFtag)
- In one case the TF was cleaved but a fraction of the cleaved TF remained assosciated with the protein. Upon diluting the sample and re-binding to Ni2+ beads followed by SEC, the TF could be separated.
apart from some of the pitfalls described above, I think it is an interesting system to explore; if you have anything soluble you may get much more with this system and it may help to get soluble proteins that you otherwise would not get.
If I receive additional comments from my structural biologist-colleagues I will update the info
just wanted to comment shortly on TF. You can see in the 3D structure of TF that the C-term is located between two domains of the protein and maybe therefore difficult to access for the protease. It could also be a problem for the fusion partner which could sterically inhibit the proper folding of TF during expression (although in solution things can be more flexible and dynamic than the static crystal structure suggests). One could design an optimized linker for this region taking the location of the C-term into account (TEV-site & strepII-tag should be long enough for the fusion partner to fit but maybe for the TEV protease the distance needs to be longer).
Could I get hold of your adapted pcoldTF vector?
Dr David I Roper
Director of Undergraduate Admissions &
Associate Professor of Structural Biology
School of Life Sciences
University of Warwick
Gibbet Hill Road
Tel 44 (0)24 7652 8369
Fax 44 (0)24 7652 3701