title: A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.
abstract: This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His₆-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹⁰ and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore. Electronic address: mdclow@nus.edu.sg.
authors.forename: Oi Wah
authors.initials: OW
authors.lastname: Liew
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
authors.forename: Cui Xia
authors.initials: CX
authors.lastname: Ang
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
authors.forename: Yu Pei
authors.initials: YP
authors.lastname: Peh
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
authors.forename: Pek Ching Jenny
authors.initials: PC
authors.lastname: Chong
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
authors.forename: Yan Xia
authors.initials: YX
authors.lastname: Ng
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore.
authors.forename: Le-Ann
authors.initials: LA
authors.lastname: Hwang
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore.
authors.forename: Xin Yu
authors.initials: XY
authors.lastname: Koh
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore.
authors.forename: Yin Mun
authors.initials: YM
authors.lastname: Yip
authors.affiliation: Thermo Fisher Scientific Inc., 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
authors.forename: A Mark
authors.initials: AM
authors.lastname: Richards
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D003346:Corticotropin-Releasing Hormone / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007114:Immunization; D007267:Injections; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D011489:Protein Denaturation; D011498:Protein Precursors / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism; D054832:Urocortins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C426705:UCN2 protein, human; D054832:Urocortins; D006639:Histidine; D003346:Corticotropin-Releasing Hormone; D020860:Subtilisin
keywords: Affinity tag removal; His(6) tag; SUMO; Thioredoxin; Urocortin 2; eXact tag