OVEREXPRESSION IN ESCHERICHIA-COLI, FOLDING, PURIFICATION, AND CHARACTERIZATION OF THE FIRST 3 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 rec eptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusi on bodies. The oligomer was recovered from solubilized inclusion bodie s using batch adsorption on SP-Sepharose. The oligomer was folded by o ne-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 m M 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 combin ation of ultrafiltration, ammonium sulfate treatment, hydrophobic inte raction, and size-exclusion chromatography. The yield of folded materi al varied between 6 and 15 mg/liter culture. The oxidation states of t he 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 methodo logy confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a sing le sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted mol ecular weight, from amino acid composition, of 21,817. The hydrodynami c properties of the molecule indicate that it is asymmetric with an ax ial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) ha s 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. (C) 19 95 Academic Press, Inc.