Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli.
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 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.
Adprotech Ltd., Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK.
Jennifer
J
White
Petra
P
Lukacik
Dirk
D
Esser
Michael
M
Steward
Naomi
N
Giddings
Jeremy R
JR
Bright
Sarah J
SJ
Fritchley
B Paul
BP
Morgan
Susan M
SM
Lea
Geoffrey P
GP
Smith
Richard A G
RA
Smith
D000595:Amino Acid Sequence; D000818:Animals; D018958:CD55 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003167:Complement Activation / Q000187:drug effects; D015926:Complement C3a / Q000037:antagonists & inhibitors; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D006168:Guinea Pigs; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
D018958:CD55 Antigens; D011994:Recombinant Proteins; D015926:Complement C3a