Production of biologically active, heterodimeric porcine interleukin-12 using a monocistronic baculoviral expression system.
A baculoviral expression system for the production of biologically active, heterodimeric interleukin (IL)-12 was developed by utilizing foot-and-mouth disease virus (FMDV) self-cleaving peptide, 2A. Recombinant porcine IL-12 (rpoIL-12) was produced by insect cells after infection with recombinant baculoviruses expressing the gene encoding a fusion protein of p35 and p40 subunits of IL-12 connected with 2A. By reducing and non-reducing SDS-PAGE analyses, it was demonstrated that rpoIL-12 had a heterodimeric structure which was resulted from 2A-dependent cleavage of the precursor fusion protein. In contrast, uncleaved, monomeric rpoIL-12 was produced by infection with baculoviruses expressing the gene lacking the 2A sequence. To assess the biological activities of these recombinants, we performed the proliferation assays of PHA-activated human PBMCs. The heterodimeric rpoIL-12 induced proliferation in a dose-dependent manner, whereas the uncleaved rpoIL-12 did not. Moreover, such biological activity was specifically inhibited by addition of anti-IL-12 antibodies or rpoIL-12 p40. These observations suggest that FMDV 2A can exert its self-cleaving activity even in a heterologous system, and that biologically active, heterodimeric rpoIL-12 can be generated by monocistronic expression of the p35/p40 fusion gene in combination with the 2A sequence.
Kokuho
T
T
Department of Biological Product, National Institute of Animal Health, Tsukuba, Ibaraki, Japan. takehiro@niah.affrc.go.jp
Watanabe
S
S
Yokomizo
Y
Y
Inumaru
S
S
D000595:Amino Acid Sequence; D000818:Animals; D005537:Aphthovirus / Q000235:genetics; D016367:Baculoviridae; D019281:Dimerization; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis; D013552:Swine / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D018664:Interleukin-12