Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker.
Journal Article (Journal Article)
Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.
Full Text
Duke Authors
Cited Authors
- Feeney, B; Soderblom, EJ; Goshe, MB; Clark, AC
Published Date
- May 2006
Published In
Volume / Issue
- 47 / 1
Start / End Page
- 311 - 318
PubMed ID
- 16289916
Pubmed Central ID
- PMC3110655
International Standard Serial Number (ISSN)
- 1046-5928
Digital Object Identifier (DOI)
- 10.1016/j.pep.2005.10.005
Language
- eng
Conference Location
- United States