Molecular cloning, expression and characterization of secreted ferritin in the silkworm, Bombyx mori

Biometals : an International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine
Yang ZhouHai-Feng Shi

Abstract

Ferritin is a ubiquitous iron storage protein which plays key role in regulating iron homeostasis and metabolism. In this paper, the ferritin heavy chain homologs (HCH) and light chain homologs (LCH) from Bombyx mori (BmFerHCH and BmFerLCH) were amplified through PCR and cloned into the expression vector pET-30a(+). The recombinant BmFerHCH and BmFerLCH expressed in Escherichia coli were in the form of insoluble inclusion bodies, indicating that the two proteins were not in their natural structural conformation. In order to obtain refolded ferritin in vitro, the inclusion bodies (BmFerHCH and/or BmFerLCH) were dissolved in denaturing buffer (100 mM Tris, 50 mM Glycine, 8 M urea, 5 mM DTT, pH 8.0) and then refolded in refolding buffer (100 mM Tris, 400 mM L-arginine, 0.2 mM PMSF, 0.5 mM DTT). The result showed that it was only when both BmFerHCH and BmFerLCH were present together in the denaturing buffer that refolding was successful and resulted in the formation of heteropolymers (H-L chain dimers) over homopolymers (H-H chain or L-L chain dimers). Moreover, the molecules (NaCl, Triton and glycerol) were found to enhance protein refolding. The optimum temperature, pH and ratios of BmFerHCH/BmFerLCH required for refolding were f...Continue Reading

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Datasets Mentioned

BETA
AK378006.1
DQ898220.1
DQ898220

Methods Mentioned

BETA
PCR
glycosylation
immunoprecipitation
Co-IP

Software Mentioned

MASCOT

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