Enhanced protein expression in mammalian cells using engineered SUMO fusions: Secreted phospholipase A₂

Abstract

SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic posttranslational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A 2 proteins (sPLA 2 ) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA 2 homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43-and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.Keywords: SUMO; SUMO fusion; phospholipase A 2 ; mammalian expression; deSUMOylase SUMO (Small Ubiquitin-like MOdifier) and the SUMO pathway are highly conserved throughout eukaryotes. SUMOylation of proteins in humans was discovered recently by Blobel and colleagues in studies of protein compartmentalization (Matunis et al. 1996). Although SUMO has low (18%) sequence identity with ubiquitin, the two proteins are very similar structurally. The SUMO and ubiquitin pathways also share many features, for instance, both proteins are synthesized as precursors requiring a proteolytic processing step prior to target protein conjugation, and each has a series of activating, conjugating, and ligating enzymes that attach the C termini of the respective proteins to the e-amino group of lysines in the target proteins (Johnson 2004). SUMO and ubiquitin hydrolases both play important regulatory roles by their removal of SUMO or ubiquitin from target proteins; however, the mechanistic similarity ends at the fate of the modified protein. Unlike ubiquitin, SUMO is not a signal for proteolysis by the 26S proteasome. Although SUMOylation is recognized as an important regulatory modification, its precise role is still the subject of active study. Several papers implicate SUMOylation as a translocation signal for nuclear-cytosolic trafficking. Paradoxically, lysine residues in target proteins that are SUMOylated are protected from ubiquitylation, while SUMOylation of proteins can also serve as a signal for ubiquitylation (Prudden et al. 2007). Thus, nature has evolved two pathways that have remarkably similar mechanical and biochemical features, yet have very distinct roles in cellular physiology (Johnson 2004).Fusion to SUMO has been shown to enhance the expression of recombinant proteins in Escherichia coli (Malakhov et al. 2004;Butt et al. 2005;Zuo et al. 2005;Marblestone et al. 2006;Panavas et al. 2008), while fusion to hSUMO3 led to similar enhancement in E. coli (data not shown) and Pichia pastoris (R.J. Peroutka, D. Roy, J. Strickler, and T.R. Butt, in prep.). Based on the or...

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…LifeSensors, Inc. has engineered a SUMO-based tag, SUMOstar, that has been used to enhance protein expression in yeast [56,57], insect cells [58], and mammalian cells [59]. In addition to the SUMOstar carrier protein, LifeSensors, Inc. has also developed a SUMOstar-specific protease.…”

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

“…For CFTR fusion, we utilized the SUMO* variant containing R64T and R71E mutations to prevent its cleavage by eukaryotic proteases (39). Analysis of the N-terminus of the fusion proteins by immunoblotting with anti-RGSHis 4 mAb suggested that SUMO*-CFTR fusions were intact in HEK cells ( Fig.…”

“…Small ubiquitin-like modifier (SUMO, Saccharomyces cerevisiae Smt3) and SUMOstar (SUMO*) domains have been shown to enhance folding and solubility of fused recombinant proteins (36, 37), including isolated CFTR NBDs (38). SUMO* is modified at two interfacial amino acids, R64T and R71E, rendering resistance to cleavage by intrinsic eukaryotic proteases (39). The SUMO* polypeptide can be removed from its fusion partner with specific proteases (37, 40).…”

The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility

“…Addition of a small ubiquitin-related modifier (SUMO) to proteins has been shown to enhance their solubility (44,45). To directly test our hypothesis that the insolubility of 343delT is the reason for low levels of detection with overexpression, we generated SUMO-HSPB5 fusion constructs.…”