title: Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro.
abstract: Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)
authors: Laboratoire de Biologie Moléculaire Végétale, Centre National de la Recherche Scientifique (Unité de Recherche 1178), Grenoble, France.
authors: O
authors: O
authors: Van Wuytswinkel
authors: G
authors: G
authors: Savino
authors: J F
authors: JF
authors: Briat
mesh_terms: D000595:Amino Acid Sequence; D002570:Ceruloplasmin / Q000235:genetics; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005293:Ferritins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D017353:Gene Deletion; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D018532:Pisum sativum / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011489:Protein Denaturation; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000378:metabolism / Q000502:physiology; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D018744:DNA, Plant; D046911:Macromolecular Substances; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D005293:Ferritins; D002570:Ceruloplasmin