Single-step purification and biological activity of human nerve growth factor produced from insect cells.
Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.
Buxser
S
S
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49001.
Vroegop
S
S
Decker
D
D
Hinzmann
J
J
Poorman
R
R
Thomsen
D R
DR
Stier
M
M
Abraham
I
I
Greenberg
B D
BD
Hatzenbuhler
N T
NT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology
D000911:Antibodies, Monoclonal; D009414:Nerve Growth Factors