CHARACTERIZATION OF HUMAN LACTOFERRIN PRODUCED IN THE BACULOVIRUS EXPRESSION SYSTEM

Lactoferrin, an iron-binding 80-kDa glycoprotein, is a major component of human milk whose structure is now well defined. The binding site o f lactoferrin to the membrane receptor of lymphocyte has been located in the region 4-52, but the amino acids directly involved in the inter action have not been identified yet. To gain further insights into the structure-function relationships of the lactoferrin binding site, we first expressed the cDNA encoding human lactoferrin in the lepidoptera Spodoptera frugiperda cells (Sf9) using a recombinant baculovirus. Th e selected transformant secreted an N-glycosylated protein of 78 kDa w hich was immunoprecipitated by specific anti-lactoferrin antibodies. T o confirm the structure and the function of the recombinant lactoferri n, the protein was purified by ion-exchange chromatography and its phy sical, biochemical, and biological properties were compared with those of the native protein. In particular, the N-terminal amino acid seque nce and the iron-binding stability as a function of pH, of both protei ns, were identical. The main difference concerns the glycosylation whi ch leads to glycans of lower molecular masses as detected by the elect rophoretic mobility of lactoferrin after N-glycosidase F treatment and matrix-assisted laser desorption ionization/time-of-flight mass spect rometry. Despite the different glycosylation features, the recombinant lactoferrin retained the binding property to the Jurkat human lymphob lastic T-cell line of the native lactoferrin. On the basis of these an alyses, production of protein mutants generated by site-directed mutag enesis is now in process. (C) 1997 Academic Press.