Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli.
The production and characterization of recombinant mouse H- and L-ferritin chains from Escherichia coli are described. The proteins were efficiently expressed and purified with yields of 7-40 mg per liter of cell culture. They had the expected molecular mass and showed a physical stability analogous to that of the corresponding human ferritins. Mouse H- and L-ferritins had a very similar mobility on denaturing SDS-PAGE, but could be readily separated on nondenaturing PAGE because of the distinct slow mobility of mouse L-ferritin. Direct comparative experiments showed that mouse and human H-ferritins had the same iron incorporation activity, whereas mouse L-ferritin incorporated iron less efficiently than human L-ferritin. The difference was attributed to the substitution of a residue exposed on the cavity surface (Glu140 --> Lys) in mouse L-ferritin, a hypothesis confirmed by the finding that the mouse L-ferritin mutant Lys140-Glu incorporated iron as efficiently as human L-ferritin. Rabbit antisera elicited by the recombinant mouse ferritins were specific for the H- and L-chains and did not cross-react with the human ferritins. The antibodies and the derived specific ELISA assays allow the determination of H- and L-ferritins in mouse tissues.
Santambrogio
P
P
Protein Engineering Unit, Department of Biological and Technological Research (Dibit), Institute H. San Raffaele, Milan, Italy.
Cozzi
A
A
Levi
S
S
Rovida
E
E
Magni
F
F
Albertini
A
A
Arosio
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000918:Antibody Specificity; D001052:Apoferritins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005293:Ferritins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D007106:Immune Sera; D011994:Recombinant Proteins; D005293:Ferritins; D001052:Apoferritins; D007501:Iron