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Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene
Zhi-Yong He1, Guan-Zhen Yang, Wei-Jie Zhang
1Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. xfwu@sunm.shcnc.ac.cn
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 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.
Insights
Researchers identified a mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor (OPG/OCIF) gene in human liver cells. This mutated OPG/OCIF isoform was successfully expressed in yeast, offering potential for therapeutic applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Osteoprotegerin/osteoclastogenesis Inhibitory Factor (OPG/OCIF) plays a crucial role in bone metabolism and regulation of osteoclastogenesis.
- Understanding OPG/OCIF gene mutations is vital for investigating related diseases and developing targeted therapies.
Purpose of the Study:
- To identify and characterize mutations in the OPG/OCIF gene in human liver cells.
- To develop a method for high-level expression of the mutated OPG/OCIF isoform in a yeast system.
Main Methods:
- Isolation of total RNA from human liver cell line L02.
- RT-PCR amplification and sequencing of OPG/OCIF cDNA.
- Cloning of the OPG/OCIF gene fragment into expression plasmids (pBS-sk and pPIC3.5K).
- Transformation of Pichia pastoris GS115 and induction of protein expression.
- SDS-PAGE analysis to assess protein expression levels and glycosylation.
Main Results:
- A novel nonsense mutation (Ochre) was identified at Gln(394) in the OPG/OCIF cDNA from L02 cells.
- The resulting OPG/OCIF isoform was 8 amino acid residues shorter at the C-terminus compared to the reported form.
- Genomic DNA sequence analysis confirmed the mutation.
- High-level expression of the mutated OPG/OCIF isoform was achieved in Pichia pastoris, accumulating to over 30% of soluble yeast protein.
- Expression levels and glycosylation increased with longer induction periods.
Conclusions:
- The study successfully identified and characterized a mutated OPG/OCIF gene in human liver cells.
- A robust yeast expression system for the mutated OPG/OCIF isoform was established.
- The findings provide a foundation for further research into the functional implications of this OPG/OCIF mutation and its potential therapeutic utility.