Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO), an active immunotherapeutic vaccine component.
The active vaccine component recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 degrees C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS-PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS-PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with pI values in the pH-range 7.0-8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 degrees C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible.
Xu
Bingze
B
Resistentia Pharmaceuticals AB, Kungsängsvägen 25, Box 853, SE-751 08 Uppsala, Sweden. Bingze.xu@astrazeneca.com
Lundgren
Mats
M
Magnusson
Ann-Christine
AC
Fuentes
Alexis
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity / Q000517:prevention & control; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009893:Opossums / Q000235:genetics*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014612:Vaccines / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D014612:Vaccines; D007073:Immunoglobulin E