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 varie ty of proteins and peptides that have diverse roles in epidermal physiology . In this report, we present purification and partial amino acid sequence o f LEKTI, a serine proteinase inhibitor, and DAN (NO3) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte condit ioned medium. Epidermal keratinocytes. were isolated from human foreskin an d serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's. modified E agle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 d ays. 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 w ere purified from heparin column passthrough by the combination of preparat ive and analytical FPLC-based gel filtration chromatography and reversed-ph ase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edma n degradation in a gas-phase sequencing system.