Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.
A cDNA construct (approximately 1 kb) of human BM-40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1-kb BM-40 mRNA and no or only little endogenous 2.2-kb mRNA. These clones also secreted BM-40 at high rates (5-50 micrograms ml-1 day-1) into serum-free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM-40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM-40 was purified by anion-exchange chromatography and showed the expected N-terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM-40 purified from the mouse Engelbreth-Holm-Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM-40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM-40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross-reaction was, however, detected between human and mouse BM-40.
Nischt
R
R
Department of Dermatology, University of Munich, Federal Republic of Germany.
Pottgiesser
J
J
Krieg
T
T
Mayer
U
U
Aumailley
M
M
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D003094:Collagen / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015676:Osteonectin / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D015676:Osteonectin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003094:Collagen; D004247:DNA; D008715:Methionine