Mammalian expression vectors – Protein Expression and Purification Core Facility

Overview of our mammalian expression vectors

For transient expression of single proteins in mammalian cells, we mainly use the pXLG vector, which was a kind gift from David Hacker from the EPFL (Switzerland).

We also have a collection of mammalian expression vectors specifically adapted for ligation-independent cloning. These vectors are called pCoofy vectors and were developed by Sabine Suppmann at the Max Planck Institute for Biochemistry in Martinsried, Munich.

The detailed cloning protocols and descriptions of these vectors can be found in the following reference:

Scholz J., Besir H., Strasser S. and Suppmann S. (2013) A new method to customize protein expression vectors for fast, efficient and background free parallel cloning. BMC Biotechnology. 13:12

Internal EMBL users can obtain these vectors at the Protein Expression and Purification Core Facility. External users can request them via Addgene.

Mammalian pCOOFY expression vectors

Name	N-terminal tag	C-terminal tag	Antibiotic resistance	Backbone
pCoofy47	VEGFss-3C	* His₁₀ * TwinStrepII * S-tag * CBP	Ampicillin	pTT5
pCoofy50	HA-His₈-Flag-3C	His₁₀	Ampicillin	pEF-HA
pCoofy55	His₁₀-SumoStar-3C	* His₁₀ * TwinStrepII * S-tag	Ampicillin	pTT5
pCoofy56	TwinStrepII-3C	His₁₀	Ampicillin	pEF-HA
pCoofy57	VEGFss-His₈-3C	* His₁₀ * TwinStrepII * S-tag * CBP	Ampicillin	pTT5
pCoofy58	VEGFss-TwinStrepII-3C	* His₁₀ * TwinStrepII * S-tag * CBP	Ampicillin	pTT5
pCoofy62	VEGFss-His₆-SumoStar	* none * His₁₀ * TwinStrepII * EPEA-tag	Ampicillin	pTT5

* Multiple options for the C-terminal tag depending on the cloning strategy

diagram — Vector map of the mammalian expression vector pXLG (David Hacker, EPFL Switzerland).

pTT5 backbone:

derived from the pTT vectors from Yves Durocher (Durocher et al., 2002, NAR)
CMV promoter

pEF-HA backbone:

2 x HA N-terminal on the backbone
EF1a promoter

For baculovirus-mediated expression in mammalian cells, we have a collection of BacMam vectors adapted for ligation-independent cloning available. These vectors are all based on the pHTBV1.1 backbone developed by Frederick Boyce from Massachusetts General Hospital, Harvard Medical School in Boston. The LIC-adapted pHBTV1.1 vectors were a kind gift from Nicola Burgess-Brown from the Structural Genomics Consortium in Oxford.

More information about the pHTBV1.1 vector can be found in the following reference:

Fornwald J.A., Lu Q., Boyce F.M. and Ames R.S.(2016) Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System. Methods Mol Biol.1350:95-116.

For the baculovirus-mediated expression of protein complexes in mammalian cells, we have the MultiBacMam vectors available. More information about how to use the MultiBacMam system can be found on the website of Geneva Biotech.