Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in Saccharomyces cerevisiae.
Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe(2+), the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.
Seo
H-Y
HY
Faculty of Biological Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, 561-756 Chonju, Republic of Korea.
Chung
Y-J
YJ
Kim
S-J
SJ
Park
C-U
CU
Kim
K-S
KS
D000818:Animals; D001052:Apoferritins; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005293:Ferritins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007501:Iron / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic
D004271:DNA, Fungal; D011994:Recombinant Proteins; D005293:Ferritins; D001052:Apoferritins; D007501:Iron