Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae.
Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2(-) and ura3(-) for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 micro g per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.
Kim
Hye-Jin
HJ
Institute for Molecular Biology and Genetics. Basic Science Research Institute, Chonbuk National University, Chonju, Korea.
Kim
Hyang-Mi
HM
Kim
Ji-Hye
JH
Ryu
Kyeong-Seon
KS
Park
Seung-Moon
SM
Jahng
Kwang-Yeup
KY
Yang
Moon-Sik
MS
Kim
Dae-Hyuk
DH
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism; D001052:Apoferritins / Q000378:metabolism; D001483:Base Sequence; D005293:Ferritins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011108:Polymers / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D014170:Transformation, Genetic
D011108:Polymers; D011994:Recombinant Proteins; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D010832:6-Phytase