A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized.
A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.
Newkirk
M M
MM
Microbiology Department, University of Texas Health Sciences Center, Dallas 75235.
Edmundson
A
A
Wistar
R
R
Klapper
D G
DG
Capra
J D
JD
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007075:Immunoglobulin M / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D012217:Rheumatoid Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D012217:Rheumatoid Factor; D010206:Papain; D003545:Cysteine