Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium.
Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO3) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.
Ahmed
A
A
Organogenesis Inc. Research & Development, Canton, Massachusetts 02021, USA. aahmed@organo.com
Kandola
P
P
Ziada
G
G
Parenteau
N
N
D000595:Amino Acid Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry* / Q000166:cytology; D008969:Molecular Sequence Data; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017077:Culture Media, Conditioned; C490087:NBL1 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors