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Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

¹ Adprotech, Ltd., Saffron Walden, Essex CB10 1XL, United Kingdom
² Laboratory of Molecular Biophysics, Biochemistry Department, Oxford University, Oxford OX1 3QU, United Kingdom
³ Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom

(RECEIVED September 25, 2003; FINAL REVISION May 7, 2004; ACCEPTED May 18, 2004)

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Keywords: complement; decay-acceleration; membrane anchor; crystallography

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate • GPI, glycophosphatidyl inositol • PpDAF, human DAF1–4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag • EcDAF, nonglycosylated human DAF 1–4 expressed in Escherichia coli • nDAF, human native glycosylated (GPI-anchored) DAF from erythrocytes • EcDAF-MP, soluble E. coli human DAF linked through a C-terminal cysteine to the myristoylated peptide APT542 • PCR, polymerase chain reaction • SCR, short consensus repeat • TCEP, Tris-(2-carboxyethyl) phosphine

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03455604.

Reprint requests to: Richard A.G. Smith, Adprotech Ltd., Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK; e-mail: r.a.smith{at}adpro.co.uk; fax: +(44) (0) 1799 532543.

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