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

Content Provider	Scilit
Author	Nischt, Roswitha Pottgiesser, Jutta Krieg, Thomas Mayer, Ulrike Aumailley, Monique Timpl, Rupert
Copyright Year	1991
Description	Journal: Journal of Biological Inorganic Chemistry 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.
Ending Page	536
Starting Page	529
ISSN	2573508X
e-ISSN	14321327
DOI	10.1111/j.1432-1033.1991.tb16214.x
Journal	Journal of Biological Inorganic Chemistry
Issue Number	2
Volume Number	200
Language	English
Publisher	Wiley-Blackwell
Publisher Date	1991-09-01
Access Restriction	Open
Subject Keyword	Journal: Journal of Biological Inorganic Chemistry Analytical Chemistry Cell Culture Human Bm Human Cell
Content Type	Text
Resource Type	Article
Subject	Biochemistry Inorganic Chemistry

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