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


  • eng

Conference Location

  • United States