Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene.
Total RNA was isolated from normal Chinese human liver cell line L02. A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid. Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394). The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported. The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned. Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA. The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115. SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days. The longer the transformant was induced, the higher the ratio of glycosylated protein was.
He
Zhi-Yong
ZY
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. xfwu@sunm.shcnc.ac.cn
Yang
Guan-Zhen
GZ
Zhang
Wei-Jie
WJ
Wu
Xiang-Fu
XF