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Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system
Authors:Michael W. Finn  Carol Clayberger  Alan M. Krensky
Affiliation:1. Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA;2. Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA;3. Soybean Genetics Improvement Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA;4. Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea;5. Department of Pediatrics, University of Maryland, School of Medicine, Baltimore, MD 21794, USA;1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China;2. Faculty of Animal Husbandry and Veterinary Medicine, Bacgiang Agriculture and Forestry University, Vietyen District, Bacgiang Province, Viet Nam;1. Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia;2. Division of Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, D-23845 Borstel, Germany;3. Department of Biotechnology, Hamburg University of Applied Science, D-21033 Hamburg, Germany;2. Department of Veterinary Pathobiology, Texas A & M University, College Station 77843-4467;1. Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA;2. Animal Bioscience & Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA;1. State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China;2. Shanghai Gebaide Biotechnical Co., Ltd., Shanghai 201403, China
Abstract:Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.
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