A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells.
Interleukin-12 (IL-12) comprises of p40 and p35 subunits that are encoded by genes on separate chromosomes and form p70 heterodimer, a bioactive protein, and free p40, an antagonist of IL-12. Balance expression of two subunits within cells would be the key for high-level of production of bioactive IL-12. Thinking about different expression efficiencies of two genes (p40 gene with higher efficiency), we selected two expression vectors with different efficiencies and inserted genes of p40 and p35 into them separately and co-transfected them into Chinese hamster ovary (CHO) cells. The high-level expression of IL-12 was obtained when p40 cDNA was inserted into pcDNA3 (lower expression vector) and p35 cDNA was inserted into pEE14 (higher expression vector), but using pEE14 for p40 cDNA and pcDNA3 for p35 cDNA, which was opposite to the optimal design, or pEE14 or pcDNA3 for both p35 cDNA and p40 cDNA did not obtain high-level of production of p70 heterodimer, the bioactive IL-12. We also observed that using two chemical reagents in combination, as a pressure selection method or amplification for the two vectors, markedly enhanced the IL-12 production, when compared with any one selection chemical. Our results indicated that the balance expressions of two chains of hetrodimer protein, such as p40 and p35 of IL-12, would be a better choice to obtain high-level of production of the proteins.
Liu
Wentao
W
Anhui Provincial Laboratory of Molecule Medicine, School of Life Sciences, University of Science and Technology of China, Hefei City, Anhui 230027, China.
Wei
Haiming
H
Liang
Shujuan
S
Zhang
Jian
J
Sun
Rui
R
Tian
Zhigang
Z
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D007962:Leukocytes / Q000378:metabolism; D021122:Protein Subunits; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018664:Interleukin-12; D007371:Interferon-gamma