Human Keratinocyte Growth Factor Recombinantly Expressed in Chinese Hamster Ovary Cells: Isolation of Isoforms and Characterization of Post-Translational Modifications
| Autor: | Yueh-Rong Hsu, Morris Charles F, Viswanatham Katta, Sylvia Hu, Julia Tseng, David W. Brankow, Hsu Eric W, Hsieng Sen Lu, William C. Kenney |
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| Rok vydání: | 1998 |
| Předmět: |
Fibroblast Growth Factor 7
Glycosylation Blotting Western CHO Cells Biology Polymerase Chain Reaction Mass Spectrometry Amidohydrolases Cell Line law.invention Mice chemistry.chemical_compound Isomerism law Cricetinae Complementary DNA Animals Humans Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Amino Acid Sequence Growth Substances Peptide sequence Chromatography High Pressure Liquid DNA Primers Glycoproteins Base Sequence Chinese hamster ovary cell Serine Endopeptidases Peptide Fragments Recombinant Proteins Fibroblast Growth Factors Carbohydrate Sequence chemistry Biochemistry Cell culture Recombinant DNA Keratinocyte growth factor Fibroblast Growth Factor 10 Protein Processing Post-Translational Biotechnology |
| Zdroj: | Protein Expression and Purification. 12:189-200 |
| ISSN: | 1046-5928 |
| Popis: | Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes. |
| Databáze: | OpenAIRE |
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