Serum transferrins recombinant

Ser Se isotopic substitution, 38 105-107 Serum albumin, 46 470 Serum transferrins, 41 390 biological role, 41 391-392 half-molecules, 41 396 recombinant, 41 453 structure, 41 397... 

Since that time many more sequences have become available through the advent of recombinant DNA technology and the deduction of amino acid sequences from the base sequences of cloned DNA. At the present time, the primary structures (amino acid sequences) of 14 proteins of the transferrin family have been established. These include seven serum transferrins, from human 10, 36), pig (37), horse 38), rabbit 39), toad Xenopus laevis) 40), sphinx moth (M. sexta) 13), and cockroach Blaberus discoidalis 4) chicken 34, 35) and duck 41) ovotransfer-rins four lactoferrins, from human (11, 42), mouse 43), pig 44) and cattle 45, 46) and the human tumor cell melanotransferrin 47). All of these sequences are available from sequence databases such as EMBL and SWISSPROT. 

Complementing the structural studies of the intact transferrins, a number of fragments have also been crystallized, including proteolytic N-terminal half-molecules of rabbit serum transferrin (69) and chicken ovotransferrin (70), recombinant N-terminal half-molecules of human lactoferrin (71) and human serum transferrin (72), and a quarter-molecule fragment of duck ovotransferrin (73). All of these have now led to high-resolution structures (74-77). 

Coordinates for human lactoferrin, in both diferric (78) and apo- (80) forms, for diferric rabbit serum transferrin (68), and for the three fragment structures, the proteolytic N-lobe of rabbit serum transferrin (74), the recombinant N-lobe of human lactoferrin (75) and the duck ovotransferrin quarter-molecule (76), all in their iron-bound forms, can be obtained from the Brookhaven Protein Data Bank (Brookhaven National Laboratory, Upton, New York). 

Where chemical or physical differences can be detected between the two sites, there remains the problem of distinguishing which site is which. For serum transferrin this is helped immensely by the ability to prepare monoferric forms, loaded in either the N- or C-site (198, 200), and to be able to separate them by electrophoresis in 6 Ilf urea, the Makey-Seal method (201). This enabled the so-called A and B sites, differentiated in earlier studies, to be identified with the C- and N-terminal sites, respectively (202). Comparisons of the diferric proteins with N- and C-loaded monoferric transferrins or (more recently) recombinant half-molecules have by now revealed a number of inequivalences. 

Fig. 28. The pH dependence of iron release from human serum transferrin (Tf), human lactoferrin (Lf), and the recombinant N-terminal half-molecule of human lactoferrin (Lfm). Also shown is a plot (dashed line) for the release of cerium from Ce4+-substituted lactoferrin, demonstrating the increased difference between the two sites for metal ions other than Fe3+.

Until now, the characterization of glycoproteins and the accompanying sample preparation have proven troublesome. Taking human serum transferrin (HST) and recombinant erythropoietin (rEPO) as examples, a possible strategy for determining oligosaccharide structures is shown below. 

Special applications of CIEF have focused on studies of glycoproteins, antibodies, and proteins in serum (e.g., hemoglobin variants [30,37,39,77-90], transferrin forms [21,31,36,80,82,90,91]), and cerebrospinal fluid [92], The gly-coforms of recombinant human tissue-type plasminogen activator [93-95] and... 

Fig. 7 Serum concentration-time course (mean SD) of erythropoietin (c) and its effect on the concentrations of free ferritin (b) and soluble transferrin receptor (a) after repeated subcutaneous administration of 200 U/kg recombinant erythropoietin in athletes (n = 18) (from [83]).

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