Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.
We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains of E. coli. Typically 2-6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.
Raffaï
R
R
Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario, K1Y 4E9, Canada.
Vukmirica
J
J
Weisgraber
K H
KH
Rassart
E
E
Innerarity
T L
TL
Milne
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000918:Antibody Specificity; D001057:Apolipoproteins E / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D066298:In Vitro Techniques; D051379:Mice; D011973:Receptors, LDL / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; D017931:DNA Primers; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011973:Receptors, LDL