Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1.
We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.
Dodd
I
I
Department of Protein Chemistry, SmithKline Beecham Pharmaceuticals, Epsom, Surrey, United Kingdom.
Mossakowska
D E
DE
Camilleri
P
P
Haran
M
M
Hensley
P
P
Lawlor
E J
EJ
McBay
D L
DL
Pindar
W
W
Smith
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D017463:Receptors, Complement 3b / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D012685:Sepharose; D012756:Sheep; D014461:Ultracentrifugation
D010446:Peptide Fragments; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins; C097574:sepharose fast flow SP; D012685:Sepharose