Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.
Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.
Ullman
C G
CG
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London, UK.
Chamberlain
D
D
Ansari
A
A
Emery
V C
VC
Haris
P I
PI
Sim
R B
RB
Perkins
S J
SJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017244:Complement Factor I / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D009036:Moths / Q000166:cytology; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
D011994:Recombinant Proteins; D017244:Complement Factor I