Recombinant expression and properties of the human calcium‐binding extracellular matrix protein BM‐40

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Abstract
A cDNA construct (∼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 μg ml‐1 day1) 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.