A self-cleavable sortase fusion for one-step purification of free recombinant proteins.
A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr-Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal His6 tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography.
Mao
Hongyuan
H
Ansata Therapeutics, Inc., Advanced Protein Labs, 505 Coast Boulevard, South, La Jolla, CA 92037, USA. hmao@san.rr.com
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003546:Cysteine Endopeptidases; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D013211:Staphylococcus aureus / Q000201:enzymology
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases