Expression in Escherichia coli of a secreted invertebrate ferritin

Abstract

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 "C and at an isopropyl P-D-thiogalactopyranoside concentration of 50 pM, 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 pg yolk ferritidl 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-aminoacid-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.Iron is an essential element for virtually all forms of life, being a constituent of many proteins involved in fundamental processes such as respiration, photosynthesis, nitrogen fixation, DNA synthesis and cell division. Sequestration of iron in a soluble, nontoxic and bio-available form is achieved by ferritins, a class of proteins which are widely distributed among prokaryotes and eukaryotes and which are capable of storing up to 4500 iron atoms/molecule inside their protein coats. Structure, function and regulation of ferritins have been intensively investigated (reviewed by Theil, 1987 ;Harrison et al., 1989Harrison et al., , 1991 Andrews et al., 1992). X-ray crystallographic analyses of both naturally occurring ferritins from vertebrates and recombinant ferritins from vertebrates and an invertebrate species revealed a remarkable conservation of three-dimensional structure (Harrison et al. 1991). Recent results from sequencing of plant ferritin cDNAs and computerassisted model-building sugge...

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

“…Another possible reason discussed further in this review may relate to the prevention of "oxidative stress." The discovery of these new IREs confirms their early evolutionary origin, which can be traced back to arthropods and mollusks (22,23). This corroborates previous results showing the conservation of IRE-binding activity in annelids and insects as well as vertebrates, and the lack of such activity in yeast, bacteria, or plants (5,24,25).…”

Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli

“…This might be due to the reason that lower temperature reduces protein synthesis rate to a stage whereby it is closely matched to the folding rate of a soluble protein [Slabaugh et al, 1993]. Similarly, lower temperature has been shown to improve solubility of ß-lactamase [Chalmers et al, 1990], MalE-Yorkferritin [vonDarl et al, 1994], PreS2-S 0 -ß-galactosidase [Thomas and Baneyx, 1996] and hemoglobin [Weickert et al, 1997] in E. coli.…”

Prussian Blue Modified Ferritin as Peroxidase Mimetics and Its Applications in Biological Detection

“…24 subunits that form a hollow spherical shell of 8 nm in inner diameter which encases a core of up to 4500 Fe (III) atoms. [12][13][14] Structural features of ferritins from humans, horse, bullfrog and bacteria are all essentially the same architecture in spite of large variations in primary structure, 15 so ferritin from other organisms may have the same functions as human ferritin. Many nanoparticles, such as gold cluster 17 and platinum nanoparticles (PtNPs), 21 have been synthesized using ferritins as nano-reactors through ferritin-loading approach [16][17][18][19][20][21] and assembly-disassembly approach.…”