Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells.
To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.
Mansour
M
M
Department of Molecular Sciences, Pfizer Central Research Division, Groton, Connecticut 06340, USA.
Karmilowicz
M
M
Hawrylik
S J
SJ
Nalcerio
B
B
Angilly
J
J
Conklyn
M J
MJ
Lilly
C M
CM
Drazen
J M
JM
Lee
S E
SE
Auperin
D D
DD
De Wet
J R
JR
Cohan
V L
VL
Showell
H J
HJ
Danley
D E
DE
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006168:Guinea Pigs; D006801:Humans; D007313:Insecta / Q000821:virology*; D015848:Interleukin-5 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D015848:Interleukin-5; D011994:Recombinant Proteins