How Ferritin was first isolated and how it is isolated now
The protein ferritin is formed from two classes of subunits, H and L, in a ratio which vary in different cell types. The H-type are subunits associated with rapid uptake of iron and are predominantly from the red blood cells. Subunits that take up iron much slower are designated L-types. A third type subunit, M, is found in amphibians and is similar to the H subunit. The M-types are predominant in the liver. Once synthesized, ferritin stores iron in a form that is nontoxic for vertebrates and invertebrates, higher plants, fungi, and bacteria. It directs the vectorial transfer of hydrates Fe(II)/Fe(II) ions to a condensed ferric phase in the central cavity of the soluble protein.
In their study, Fetter et al. (1997) monitored the kinetic of formation of the Fe(III)-protein intermediates in frog H-ferritin and frog M-ferritin. They used Fe(III)-tyrosine species that absorb at A550 nm or A650- for the H-ferritin or M-ferritin, respectively. These species are chosen because their absorbance is not affected by the absorbance of other transient species that form or stable minerals that are present in the sample. The following is the mutagenesis and protein isolation procedure followed for this study:
The coding sequences for frog H and …show more content…
(1997) also isolated ferritin via protein expression of pET3 vector that contains 5F12 coding sequence in the NdeI sequence site for the H-type subunit of the frog ferritin. The vector host was Escherichia coli BL21-3 cells grown in medium containing sorbital and betaine. Ferritin synthesis was induced in mid-log cells using isothiopropyl thiogalactoside (IPTG), and the soluble ferritin was isolated, after sonication of the bacterial cells, by fast protein liquid chromatography (FPLC) on a Fast Q-Sepharose matrix. Protein purified to homogeneity was sterilized by filtration through a 0.45 µm membrane and stored at 4 °C for less than 1 week before mixing with
The protein ferritin is formed from two classes of subunits, H and L, in a ratio which vary in different cell types. The H-type are subunits associated with rapid uptake of iron and are predominantly from the red blood cells. Subunits that take up iron much slower are designated L-types. A third type subunit, M, is found in amphibians and is similar to the H subunit. The M-types are predominant in the liver. Once synthesized, ferritin stores iron in a form that is nontoxic for vertebrates and invertebrates, higher plants, fungi, and bacteria. It directs the vectorial transfer of hydrates Fe(II)/Fe(II) ions to a condensed ferric phase in the central cavity of the soluble protein.
In their study, Fetter et al. (1997) monitored the kinetic of formation of the Fe(III)-protein intermediates in frog H-ferritin and frog M-ferritin. They used Fe(III)-tyrosine species that absorb at A550 nm or A650- for the H-ferritin or M-ferritin, respectively. These species are chosen because their absorbance is not affected by the absorbance of other transient species that form or stable minerals that are present in the sample. The following is the mutagenesis and protein isolation procedure followed for this study:
The coding sequences for frog H and …show more content…
(1997) also isolated ferritin via protein expression of pET3 vector that contains 5F12 coding sequence in the NdeI sequence site for the H-type subunit of the frog ferritin. The vector host was Escherichia coli BL21-3 cells grown in medium containing sorbital and betaine. Ferritin synthesis was induced in mid-log cells using isothiopropyl thiogalactoside (IPTG), and the soluble ferritin was isolated, after sonication of the bacterial cells, by fast protein liquid chromatography (FPLC) on a Fast Q-Sepharose matrix. Protein purified to homogeneity was sterilized by filtration through a 0.45 µm membrane and stored at 4 °C for less than 1 week before mixing with