Recombinant protein production in the BacMam system
Protein purification begins with expression in cells. Different cell expression systems offer certain benefits and drawbacks to yield and post-translational modification. TriAltus has expanded our plasmid offerings to include BacMam vectors so that the CL7/Im7 affinity purification system can be used in a wider scope of applications.
What is BacMam?
BacMam is baculovirus-mediated transduction of mammalian cells. Using a virus from insect cells (baculovirus) allows for efficient gene delivery and expression in mammalian cells, including cell types that are difficult to transfect by other means, such as primary cells or stem cells. TriAltus’ BacMam vectors don’t have selective markers to establish a stable cell line, but they can be used for transient transfection in HEK293 or CHO cells, for example.
The BacMam system works by first inserting your gene of interest into the BacMam vector and using it to generate a recombinant bacmid using the Bac-to-Bac system. This bacmid is used to transfect insect cells to generate the recombinant baculovirus that can then transduce mammalian cells.
What are the benefits of BacMam?
Proteins can be expressed and purified from insect and mammalian cells separately, but BacMam combines the benefits of both options. Insect cells have high protein expression levels and allow for many of the same post-translational modifications as in mammalian cells. Insect cells also allow for the option to secrete expressed proteins into the media rather than purifying from cell lysate. Despite these benefits, some eukaryotic proteins still require expression in a mammalian cell line. BacMam bridges the gap by including mammalian transcriptional elements in a baculovirus’ DNA.
What’s in TriAltus’ BacMam vectors?
TriAltus’ BacMam vectors have both standard and unique features. Transcription is driven by a CMV promoter (human cytomegalovirus) that drives transcription in mammalian cells. In order to achieve suitable expression levels, a Kozak sequence must be included before the start codon: 5’- A/GCCATGG -3’ where ATG is the start codon. Our N-terminal tagged vector includes this sequence while the C-terminal tagged vector requires it to be added easily through your designed forward primer.
PH (polyhedron) and P10 baculovirus promoters are also included. The gene expressed by the PH promoter will be tagged, while P10 is used for co-expressing a protein if desired.
TriAltus’ BacMam vectors are unique among commercially available options in that they include three tags: CL7, His8, and eGFP. CL7 gives you the ability to purify your protein of interest using Im7 resin. His8 is a common tag for many vectors and is available for those who require double-tagging. eGFP is included for visualization purposes. All three tags are cleavable from the protein of interest using PSC protease.
BacMam NT H8-CL7-eGFP vector map
BacMam CT eGFP-CL7-H8 vector mapDownload Genbank files of the BacMam vectors from our Plasmid Guide here
BacMam is a useful tool for scientists who want to combine the benefits of insect and mammalian expression systems to produce well-expressed, post-translationally modified proteins.