Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.
Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.
Liu
Li
L
Lifesensors, Inc., Research and Development, Malvern, PA 19355, USA.
Spurrier
Joshua
J
Butt
Tauseef R
TR
Strickler
James E
JE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C486203:USP4 protein, human; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C118306:Usp18 protein, mouse; C579226:USP15 protein, human; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; C579225:Usp15 protein, mouse