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 of lactoferrin to the membrane receptor of lymphocyte has been located in the region 4-52, but the amino acids directly involved in the interaction 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. The selected transformant secreted and N-glycosylated protein of 78 kDa which was immunoprecipitated by specific anti-lactoferrin antibodies. To confirm the structure and the function of the recombinant lactoferrin, the protein was purified by ion-exchange chromatography and its physical, biochemical, and biological properties were compared with those of the native protein. In particular, the N-terminal amino acid sequence and the iron-binding stability as a function of pH, of both proteins, were identical. The main difference concerns the glycosylation which leads to glycans of lower molecular masses as detected by the electrophoretic mobility of lactoferrin after N-glycosidase F treatment and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Despite the different glycosylation features, the recombinant lactoferrin retained the binding property to the Jurkat human lymphoblastic T-cell line of the native lactoferrin. On the basis of these analyses, production of protein mutants generated by site-directed mutagenesis is now in process.
Salmon
V
V
Laboratoire de Chimie Biologique, Centre National de la Recherche Scientifique, Université des Sciences et Technologies de Lille, Villeneuve, France.
Legrand
D
D
Georges
B
B
Slomianny
M C
MC
Coddeville
B
B
Spik
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007501:Iron / Q000378:metabolism; D019169:Jurkat Cells / Q000378:metabolism; D007781:Lactoferrin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin