A. Ahmed et al., Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium, J PROTEIN C, 20(4), 2001, pp. 273-278
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.