Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin |
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Authors: | Kyung-Suk Kim |
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Institution: | (1) Faculty of Biological Sciences and Institute for Molecular Biology and Genetics, Chonbuk National University, 561-756 Chonju, Korea |
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Abstract: | In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three
mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins
were produced inEscherichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas
the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine.
Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble
ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritins were capable
of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further
functional analysis was also made on the individual amino acid residues of ferroxidase center. |
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Keywords: | mutagenesis ferritin assembly solubility |
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