Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker.
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.
Feeney
Brett
B
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
Soderblom
Erik J
EJ
Goshe
Michael B
MB
Clark
A Clay
AC
D000595:Amino Acid Sequence; D053148:Caspase 3 / Q000378:metabolism* / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D005982:Glutathione Transferase; D053148:Caspase 3