A SUMO-Groucho Q domain fusion protein: characterization and in vivo Ulp1-mediated cleavage.
We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.
Kuo
Dennis
D
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA.
Nie
Minghua
M
De Hoff
Peter
P
Chambers
Michael
M
Phillips
Martin
M
Hirsch
Ann M
AM
Courey
Albert J
AJ
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D025842:SUMO-1 Protein / Q000096:biosynthesis* / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D003546:Cysteine Endopeptidases; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D025842:SUMO-1 Protein; C091023:gro protein, Drosophila; C118083:Ulp1 protease