Membrane combined with hydrophilic macromolecules enhances protein refolding at high concentration |
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| Institution: | 1. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;2. Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China;1. Department of Physics, University of Texas at San Antonio, San Antonio, TX 78249, USA;2. Department of Biology and Neurosciences Institute, University of Texas at San Antonio, San Antonio, TX 78249, USA;1. Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Corrensstr. 48, Muenster 48149, Germany;2. Department of Pharmaceutical and Medicinal Chemistry – Clinical Pharmacy, University of Muenster, Corrensstr. 48, 48149 Muenster, Germany;3. Department of Pediatric Hematology and Oncology, University Children''s Hospital Muenster, Muenster, Germany;1. Departamento de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, IMIB-Arrixaca, 30100 Murcia, Spain;2. Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO), Carretera de la Azohía s/n, Puerto de Mazarrón, 30860 Murcia, Spain;1. Department of Biology, University of Bergen, Bergen, Norway;2. National Institute of Nutrition and Seafood Research, Bergen, Norway;1. Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain;2. Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain;3. Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Ciencias, Universidad de Santiago de Compostela, 27002 Lugo, Spain |
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| Abstract: | Membrane technology is important to the development of modern biotechnology. It has the potential to efficiently refold protein at high concentration that is still a challenge for pharmaceutical protein produced from inclusion bodies. This paper dealt with the application of a polysulfone hollow fiber membrane to protein refolding using recombinant human granulocyte colony-stimulating factor (rhG-CSF) as a model protein. Compared with dilution refolding at protein concentration of 1.0 mg/mL, the crossflow membrane system led to a 16% increase in soluble protein recovery, and a 3.3-fold increase in specific bioactivity. Addition of PEG 6 K at 2 g/L could further improve the soluble protein recovery up to 57%, the specific bioactivity up to 2.2 × 108 IU/mL. Addition of dextran at 5 g/L could increase the soluble protein recovery up to 63.6%, the specific bioactivity up to 2.30 × 108 IU/mL. By gently and gradually removing denaturant, ultrafiltration membrane system was demonstrated to be very helpful for protein refolding at high concentration. Combining with hydrophilic macromolecular of PEG or dextran could further increase its efficiency. PEG was able to promote the refolding intermediate of rhG-CSF to transfer into the native structure; whereas dextran could enhance protein refolding mainly by weakening shear stress-induced protein aggregation. |
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| Keywords: | Refolding Ultrafiltration rhG-CSF Inclusion bodies Polyethylene glycol Dextran |
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