Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.
Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants.
van Veen
Harrie A
HA
Pharming, Archimedesweg 4, 2333 CN Leiden, The Netherlands. h.veen@pharming.com
Geerts
Marlieke E J
ME
van Berkel
Patrick H C
PH
Nuijens
Jan H
JH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011110:Polymorphism, Genetic; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000596:Amino Acids; D002412:Cations; D011994:Recombinant Proteins; D007781:Lactoferrin