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

Plant ferritin subunits are synthesized as precursor molecules; the tr ansit peptide (TP) in their NH2 extremity, responsible for plastid tar geting, is cleaved during translocation to this compartment. In additi on, the N-terminus of the mature subunit contains a plant-specific seq uence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhere, Massenet, and Theil, E. C. (1990) J. Biol. Chem. 265, 18339-18344], th e 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 engineer ed 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 sequenc es deleted and (4) containing the TP but with the EP deleted. These fo ur DNA fragments have been cloned in an Escherichia coli expression ve ctor to produce the corresponding recombinant pea-seed ferritins. Expr ession 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 bet aine allowed soluble proteins to accumulate when constructs with the T P deleted were used; under this condition, E. coli cells transformed w ith constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were fo und to be assembled only when the TP was deleted; however assembled fe rritin 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 contai ning an average of 40-50 iron atoms per molecule. Despite the conserva tion 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 act ive than the recombinant human L-ferritin. The recombinant ferritin wi th 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 renaturat ion by dialysis the r Delta TP/EP protein showed a 25% increase in cor e-formation in vitro compared with the r Delta TP protein. These resul ts demonstrate (1) that the TP must be deleted to produce assembled ac tive recombinant pea-seed ferritin in E. coli and (2) that the specifi c sequence (EP) found at the N-terminus of the mature subunit of plant ferritin plays an active role in the structure/function properties of this protein, although it is not essential for obtaining assembled mo lecules active in iron-core formation.