cDNA cloning and bacterial expression of the human type I keratin 16.
The human type I keratin 16 is constitutively expressed in a number of complex epithelial tissues, including skin, but is better known for its induction under conditions favoring enhanced proliferation or abnormal differentiation, including wound healing, psoriasis, and cancer. We cloned the coding sequence of human K16 by applying a coupled reverse transcription-polymerase chain reaction procedure to mRNAs prepared from cultured human skin keratinocytes. We then expressed the human K16 coding sequence in E. coli and purified the solubilized protein by anion-exchange chromatography. The recombinant protein recovered behaves similarly to human K14 (a related acidic keratin) on the anion-exchanger, co-migrates with native human K16 on SDS-PAGE (M(r) 48 kD), and reacts with antisera directed against human K16. Based on the nucleotide sequence obtained and the properties of the corresponding recombinant protein, we conclude that we have cloned the coding portion of the human K16 cDNA. The sequence data obtained in this study is compared to earlier reports of the human K16 sequence, which are conflicting in many respects. The availability of K16 in a purified recombinant form will allow us to study how its properties may relate to its function during wound healing and in skin diseases.
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D007633:Keratins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D012867:Skin / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007633:Keratins