Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells

Lee, Geum Young; Jung, Woon Won; Kang, Chang Soo; Bang, In Seok

doi:10.1016/j.pep.2005.09.023

Cited by 20 publications

(11 citation statements)

References 34 publications

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“…Insect ovary cells (HighFive, Invitrogen) were maintained in a serum free medium (ExpressFive, Invitrogen), seeded in collagen type I-coated culture bottles (BioCoat, Becton Dickinson, CA, USA), and incubated at 28°C in a humidified room air. 20 The vector was expressed in the cells using a transfection reagent (FuGENE HD, Invitrogen), and the supernatant was harvested. Small particles were removed from the supernatant using 0.45 mm filters (MILLEX-HA, Millipore, MA, USA), and the supernatant was concentrated to ×30 using Amicon Ultra-15 filters (cut off 10 kD, Ultracel-10k, Millipore).…”

Section: Vector Expression and Hepo Purificationmentioning

confidence: 99%

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

Matsushima¹,

Toba²,

Hanawa³

et al. 2012

Drug Delivery

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Section: Vector Expression and Hepo Purificationmentioning

confidence: 99%

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

Matsushima¹,

Toba²,

Hanawa³

et al. 2012

Drug Delivery

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“…This mass range disagrees with the expected MW of the glycosylated VEGF 165 according to the literature. MW values of 46 kDa [13] and 42-44 kDa [12] have been assigned to glycosylated human VEGF 165 although based on SDS-PAGE analyses which are not accurate for mass determination. The MW of the largest glycosylated VEGF 165 form calculated according to the results shown by Keck et al [14] is 43 020 Da.…”

Section: Analysis Of the Ms Spectramentioning

confidence: 99%

“…The VEGF 165 is a glycoprotein secreted as a homodimer consisting of two 165 amino acid monomers covalently linked by two disulfide bridges. Different bibliographic sources report molecular weight (MW) in the range of 44-48 kDa for VEGF 165 [12,13]. Each VEGF 165 monomer has several intra-disulfide bridges, either three (www.expasy.org/uniprot/P15692) or seven [14] according to the bibliographic source.…”

Section: Introductionmentioning

confidence: 99%

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Development of a CE‐MS method to analyze components of the potential biomarker vascular endothelial growth factor 165

Puerta

Bergquist

2009

Electrophoresis

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The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF‐A family. Its angiogenic effect is involved in many physiological and pathological events. For this reason, its roles as a potential biomarker and as a therapeutic drug have been considered. Nevertheless, very little is known about the existence of different forms of VEGF165 arising from glycosylation and potentially from other PTMs. This aspect is important because different forms may differ in biological activity (therapeutic drug application) and the pattern of the different forms can vary with pathological changes (biomarker application). In this work a CE‐MS method to separate up to seven peaks containing, at least, 19 isoforms of intact VEGF165 is described. Comparison between human VEGF165 expressed in a glycosylating system, i.e. insect cells, and in a non‐glycosylating system, i.e. E. coli cells, has been carried out. The method developed provides structural information (mass fingerprint) about the different forms of VEGF165 and after the deconvolution and the analysis of the MS spectra, PTMs pattern of VEGF165 including glycosylation and loss of amino acids at the N‐ and C‐terminus was identified. Glycans involved in PTMs promoting different glycoforms observed in the CE‐MS fingerprint were confirmed by MALDI‐MS after deglycosylation with peptide N‐glycosidase F. This approach is a starting point to study the role of VEGF165 as a potential biomarker and to perform quality control of the drug during manufacturing. To our knowledge this is the first time that a CE‐MS method for the analysis of VEGF165 has been developed.

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“…A concentração de 100 ηg/mL foi escolhida por já ter sido utilizada em trabalhos de diferentes grupos, tendo mostrado ser suficiente para resultar em respostas biológicas (Catena et al, 2010;G. Y. Lee, Jung, Kang, & Bang, 2006;S.-B.…”

Section: Imageamento Em Tempo Real Das Culturas Tridimensionais (3d) unclassified

Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual

Belchior¹

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Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells

Cited by 20 publications

References 34 publications

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

Development of a CE‐MS method to analyze components of the potential biomarker vascular endothelial growth factor 165

Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual

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