SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins

Abstract

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.

Screen for multi-SUMO–binding proteins reveals a multi-SIM–binding mechanism for recruitment of the transcriptional regulator ZMYM2 to chromatin

“…To do this, we created a doxycycline-inducible stable cell line expressing a FLAG-tagged version of SUMO3 lacking a key lysine residue needed for polychain formation (K11R). Furthermore, to enhance our ability to detect multi-SUMOylated species, we also introduced the Q90P mutation into SUMO3, which renders it noncleavable from substrates by SENPs (31,32). As predicted, SUMO3 was detected in cells expressing SUMO3(K11R/Q90P) as a series of high-molecular-weight bands (Fig.…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…The pcpD gene from Sphingomonas sp. RA2 was amplified by PCR and cloned into a pET28 vector (Novagen) downstream of and in-frame with a His 10 -tagged Smt3 protein [small ubiquitin-related modifier (SUMO)] from Saccharomyces cerevisiae (54). The sequence of pcpD was verified by DNA sequencing.…”

“…The cells were lysed by two passages through a French Press. Nucleic acids were precipitated with 0.3% protamine sulfate, and the clarified lysate was loaded on an Ni 2+ -column (5 mL of HisTrap HP from GE Healthcare), and eluted using a linear gradient of 100-500 mM imidazole in buffer B. Fractions containing PcpD were pooled and treated with Ulp1 (SUMO protease 1) to remove the Smt3 tag using a protocol described in Malakhov et al (54). Tag-free PcpD was concentrated and applied to a Superdex 200 16/60 gel filtration column (GE Healthcare) and eluted with buffer B. Fractions containing PcpD as determined by absorption spectrum and SDS/PAGE were pooled, concentrated, flash frozen, and stored at −80°C.…”

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

“…In a high-throughput mode, fluorescent nucleotides would likely be used in place of radioactivity, which would be expected to increase the signal/noise ratio as well as to give acceptable precision. SUMO protease, shown to be a robust enzyme capable of acting under broad temperature, pH, and ionic strength ranges [38], apparently can release active polymerase from the fusion quite rapidly given that similar kinetics of incorporation was seen in the cases of precleaved 3D pol and fused 3D pol incubated simultaneously with SUMO protease and components of the polymerase reaction. Moreover, incorporation kinetics using 3D pol purified in the traditional fashion [34] was similar to that of the 3D pol -SUMO fusion cleaved with SUMO protease (Fig.…”

“…Processing of the This article describes the development of an assay for the isopeptidase enzyme SUMO protease, the catalytic domain of yeast Ulp1 [37]. This protease has been evaluated as a tool for purification of proteins from SUMO fusions expressed in E. coli and has been found to be extremely robust and precise in its cleavage activity [38]. SUMO protease activity is coupled to the activation of the 3D pol viral polymerase.…”