Cloning and Expression
of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene
HE Zhi-Yong1,2, YANG Guan-Zhen1, ZHANG
Wei-Jie2 and WU Xiang-Fu1
( 1Shanghai Institute of Biochemistry, the Chinese Academy of
Sciences, Shanghai 200031, China； 2College of Life Science and
Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China )
Abstract 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 Gln394. 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.
Key Words osteoclastogenesis inhibitory
factor；
mutated gene； cloning； Pichia pastoris； expression
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