Production of lymphotoxin (LT alpha) and a soluble dimeric form of its receptor using the baculovirus expression system.
Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.
Crowe
P D
PD
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
VanArsdale
T L
TL
Walter
B N
BN
Dahms
K M
KM
Ware
C F
CF
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002478:Cells, Cultured; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007313:Insecta; D008233:Lymphotoxin-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017924:Nucleopolyhedroviruses / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D008233:Lymphotoxin-alpha; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins