A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies
| Autor: | Yin Mun Yip, Xin Yu Koh, A. Mark Richards, Oi Wah Liew, Yan Xia Ng, Yu Pei Peh, Le-Ann Hwang, Pek Ching Jenny Chong, Cui Xia Ang, Wei Liu |
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| Rok vydání: | 2013 |
| Předmět: |
Protein Denaturation
Saccharomyces cerevisiae Proteins medicine.drug_class Corticotropin-Releasing Hormone Recombinant Fusion Proteins Immunology Molecular Sequence Data Protein tag Monoclonal antibody medicine.disease_cause Chromatography Affinity law.invention Injections Mice Affinity chromatography law Antibody Specificity Protein purification medicine Escherichia coli Immunology and Allergy Animals Humans Histidine Amino Acid Sequence Cloning Molecular Protein Precursors Urocortins Mice Inbred BALB C Chemistry Subtilisin Antibodies Monoclonal Molecular biology Biochemistry Solubility Mutation Recombinant DNA Small Ubiquitin-Related Modifier Proteins Immunization Thioredoxin Oligopeptides Myc-tag Protein Binding |
| Zdroj: | Journal of immunological methods. 403(1-2) |
| ISSN: | 1872-7905 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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