Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis |
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| Affiliation: | 1. Department of Haematology, The Queen’s University of Belfast, Royal Victoria Hospital, Belfast, Northern Ireland, UK;2. Department of Virology, Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Stoney Road, Belfast, Northern Ireland, UK;1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Provincial Key Laboratory of Applied Marine Biology (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, PR China;2. South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, PR China;1. Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea;2. Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea;3. Corea Cheju Origin Roh''s Aquariums, Jongdal-ri, Gujwa-eup, Jeju Self-Governing Province 63364, Republic of Korea;1. Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou, 515063, China;2. Institute of Marine Sciences, Shantou University, Shantou, 515063, China;3. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, 450002, China |
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| Abstract: | Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil–granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na2HPO4 buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na2HPO4 buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique. |
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