title: Human SUMO fusion systems enhance protein expression and solubility.
abstract: A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130021, China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Wang
authors.forename: Haolong
authors.initials: H
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Guan
authors.forename: Haili
authors.initials: H
authors.lastname: Ling
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Wang
authors.forename: Tianyang
authors.initials: T
authors.lastname: Mu
authors.forename: Franklin D
authors.initials: FD
authors.lastname: Shuler
authors.forename: Xuexun
authors.initials: X
authors.lastname: Fang
mesh_terms: D019677:Artificial Gene Fusion; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins