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Purification and Partial Amino Acid Sequence of Proteins from Human Epidermal Keratinocyte Conditioned Medium

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Abstract

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.

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Correspondence to Aftab Ahmed.

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Ahmed, A., Kandola, P., Ziada, G. et al. Purification and Partial Amino Acid Sequence of Proteins from Human Epidermal Keratinocyte Conditioned Medium. J Protein Chem 20, 273–278 (2001). https://doi.org/10.1023/A:1010902815953

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