Production of human secretory component with dimeric IgA binding capacity using viral expression systems.
The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.
Rindisbacher
L
L
Institut de Biologie Animale, Bâtiment de Biologie, Université de Lausanne, Switzerland.
Cottet
S
S
Wittek
R
R
Kraehenbuhl
J P
JP
Corthésy
B
B
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012635:Secretory Component / Q000096:biosynthesis* / Q000378:metabolism; D018411:Spodoptera; D014616:Vaccinia virus / Q000235:genetics
D007070:Immunoglobulin A; D011994:Recombinant Proteins; D012635:Secretory Component; C034639:polymeric IgA