Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
| Autor: | Jacquelyn V. Hawes, Eric Martin, Chaz M. Cuckler, Michael C. Bates, Charles J. Dimitroff, Monica M. Burdick, Grady E. Carlson, Steven R. Barthel, Nathan M. Reynolds |
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| Rok vydání: | 2014 |
| Předmět: |
Lysis
Galectin 1 Recombinant Fusion Proteins General Chemical Engineering Bioengineering Chromatography Affinity General Biochemistry Genetics and Molecular Biology law.invention Mice chemistry.chemical_compound Affinity chromatography law Animals Humans Staphylococcal Protein A Chromatography biology General Immunology and Microbiology General Neuroscience Immunoglobulin Fc Fragments Membranes Artificial Molecular biology Membrane Workflow chemistry Recombinant DNA biology.protein Agarose Protein A |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/51023-v |
| Popis: | Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods. |
| Databáze: | OpenAIRE |
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