Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain

Mayer, U, Pöschl, E, Nischt, R, Specks, U, Pan, T C, Chip, M L and Timpl, R (1994) Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain. European Journal of Biochemistry, 225 (2). pp. 573-80. ISSN 0014-2956

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Abstract

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Item Type: Article
Uncontrolled Keywords: amino acid sequence,aprotinin,base sequence,cell line,cells, cultured,collagen,cross reactions,dna primers,fibroblasts,fluorescent antibody technique,gene expression,humans,kidney,molecular sequence data,peptides,plant proteins,radioimmunoassay,recombinant proteins,transfection,trypsin inhibitors
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Pure Connector
Date Deposited: 13 Jan 2016 11:00
Last Modified: 11 Apr 2019 14:53
URI: https://ueaeprints.uea.ac.uk/id/eprint/56279
DOI: 10.1111/j.1432-1033.1994.00573.x

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