High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars.
Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 10(7) receptors/cell or 3 micrograms active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0.6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.
Pajot-Augy
E
E
Unité d'Ingénierie des Protéines, INRA-Biotechnologies, Jouy-en-Josas, France.
Couture
L
L
Bozon
V
V
Remy
J J
JJ
Biache
G
G
Severini
M
M
Huet
J C
JC
Pernollet
J C
JC
Salesse
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D007700:Kinetics; D007915:Lepidoptera; D008969:Molecular Sequence Data; D011974:Receptors, LH / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
D011974:Receptors, LH; D011994:Recombinant Proteins