Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO.
Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.
Haniu
M
M
Amgen, Inc., Thousand Oaks, California 91320.
Narhi
L O
LO
Arakawa
T
T
Elliott
S
S
Rohde
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry*; D004926:Escherichia coli; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin