Mammalian cell transient expression of tissue factor for the production of antigen.
We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.
Paborsky
L R
LR
Department of Cardiovascular Research, Genentech, Inc., San Francisco, CA 94080.
Fendly
B M
BM
Fisher
K L
KL
Lawn
R M
RM
Marks
B J
BJ
McCray
G
G
Tate
K M
KM
Vehar
G A
GA
Gorman
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D018139:Simplexvirus / Q000235:genetics / Q000276:immunology; D013925:Thromboplastin / Q000235:genetics* / Q000276:immunology; D014612:Vaccines; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D013925:Thromboplastin