Characterization of recombinant bovine conglutinin expressed in a mammalian cell.
We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical and biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 microg/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity.
Suzuki
Y
Y
Department of Pathology, Osaka Prefectural Institute of Public Health, Higashinari, Japan.
Eda
S
S
Kawai
T
T
Ohtani
K
K
Kase
T
T
Sakamoto
T
T
Wakamiya
N
N
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D037222:Collectins; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006385:Hemagglutination Inhibition Tests; D009980:Influenza A virus; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012712:Serum Globulins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D014170:Transformation, Genetic
D037222:Collectins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012712:Serum Globulins; C024014:conglutinin