OpenLB
Open Library of Bioscience

The coding regions of the cDNAs for cytoplasmic soma ferritin and secreted yolk ferritin from the snail Lymnaea stagnalis were inserted into the prokaryotic expression vector pEMBLex2. The vector directed the synthesis in Escherichia coli of soma ferritin up to a concentration of 15% of soluble proteins. Soma ferritin was expressed as the multimeric protein (480 kDa). Its similarity with natural soma ferritin was confirmed by PAGE, immunostaining and electron microscopy. Yolk ferritin was expressed in the form of inclusion bodies. Attempts to refold and assemble the purified yolk ferritin subunit in vitro failed. The yolk ferritin coding sequence was therefore inserted into the expression vector pMAL-p2. At a growth temperature of the bacterial cells of 23 degrees C and at an isopropyl beta-D-thiogalactopyranoside concentration of 50 microM, about 5% of the induced MalE-yolk-ferritin fusion protein was secreted into the periplasmic space and could be purified by affinity chromatography on amylose; the rest occurred as insoluble cytoplasmic inclusion bodies. Soluble MalE-yolk-ferritin fusion protein was capable of assembly into ferritin-like particles. Fully assembled yolk apoferritin shells (610 kDa) were obtained by digestion of these particles with proteinase K (yield: 180 micrograms yolk ferritin/l bacterial culture). Recombinant yolk ferritin was capable of taking up iron in vitro. Yolk ferritin (610 kDa) and soma ferritin (480 kDa) were run to the pore limit of a non-denaturing 5-20% PAGE gradient gel. Under these conditions, yolk ferritin had a higher mobility than soma ferritin (480 kDa) and therefore the yolk ferritin may have a rather compact structure. A 41-amino-acid-residue stretch of the insertion, a distinctive feature of the yolk ferritin subunit, was deleted by site-directed mutagenesis. The MalE-yolk-ferritin variant thus obtained was readily degradable by proteinase K and could not be assembled into ferritin-like particles. Therefore residues in the deleted peptide must be important for the maintenance of the native structure.