TY - JOUR
T1 - In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus
AU - Gordon, M.
AU - Chokshi, H.
AU - Alpers, D. H.
N1 - Funding Information:
This work was supportedin part by the Monsanto Company( St. Louis, MO), and by grant DK-33487 from the NationalI nstituteso f Health.T he authors thank Ms. Elsa Forero for preparationo f the manuscript.
PY - 1992/10/20
Y1 - 1992/10/20
N2 - Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 μg per 106 cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6 · 10-10 M) and for the intrinsic factor-cobalamin receptor (3.5 · 10-10 M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after clution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.
AB - Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 μg per 106 cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6 · 10-10 M) and for the intrinsic factor-cobalamin receptor (3.5 · 10-10 M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after clution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.
KW - Cobalamin binding protein
KW - Eukaryotic expression
KW - IF-cbl
UR - http://www.scopus.com/inward/record.url?scp=0026675528&partnerID=8YFLogxK
U2 - 10.1016/0167-4781(92)90161-R
DO - 10.1016/0167-4781(92)90161-R
M3 - Article
C2 - 1420307
AN - SCOPUS:0026675528
SN - 0167-4781
VL - 1132
SP - 276
EP - 283
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 3
ER -