SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.
SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides.
Malakhov
Michael P
MP
LifeSensors, Inc., 271 Great Valley Parkway, Malvern, PA 19355, USA.
Mattern
Michael R
MR
Malakhova
Oxana A
OA
Drinker
Mark
M
Weeks
Stephen D
SD
Butt
Tauseef R
TR
D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D010450:Endopeptidases; C405949:ULP2 protein, S cerevisiae; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease