Altered glycosylation and selected mutation in recombinant human complement component C9: effects on haemolytic activity

Taylor, Kathryn Mary ORCID: https://orcid.org/0000-0002-9576-9490, Morgan, Bryan Paul ORCID: https://orcid.org/0000-0003-4075-7676 and Campbell, Anthony Keith 1994. Altered glycosylation and selected mutation in recombinant human complement component C9: effects on haemolytic activity. Immunology 83 (3) , pp. 501-506.

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Abstract

Recombinant wild-type and mutated forms of human complement component C9 have been synthesized in baculovirus-infected insect cells. Wild-type recombinant C9 was indistinguishable from native C9, as judged by haemolytic activity, trypsin and alpha-thrombin digestion, reaction with antibodies to C9, enzymatic deglycosylation to the same core size and polymerization in the presence of Zn2+. Replacement of the native signal peptide with the honey-bee melittin signal peptide, and replacement of Spodoptera frugiperda (Sf9) cells with Trichoplusia ni cells produced yields of 5 micrograms C9/ml supernatant. Three C9 mutants were generated; one mutant, with four acidic residues changed to alanines in a putative calcium-binding site, had the same biological activity as recombinant C9. Another mutant, lacking 23 N-terminal amino acids, previously showing increased polymerization when produced in vitro, polymerized on secretion, rendering it inactive. It was not possible to demonstrate haemolytic activity of the third mutant, cysteines 33 and 36 mutated to alanine, as it was secreted a hundredfold less than the wild-type protein.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Pharmacy
Subjects:	Q Science > QD Chemistry Q Science > QR Microbiology R Medicine > RM Therapeutics. Pharmacology
Publisher:	Wiley
ISSN:	1365-2567
Last Modified:	19 Oct 2022 09:52
URI:	https://orca.cardiff.ac.uk/id/eprint/22492

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