Assembly of a Functional Immunoglobulin F v Fragment in Escherichia coli
0303 health sciences
03 medical and health sciences
Myeloma Proteins
Phosphorylcholine
Genetic Vectors
Escherichia coli
Immunoglobulin Variable Region
Immunoglobulin Fragments
Recombinant Proteins
Plasmids
3. Good health
DOI:
10.1126/science.3285470
Publication Date:
2006-10-05T17:21:15Z
AUTHORS (2)
ABSTRACT
An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in
Escherichia coli
. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the F
v
fragment in
E. coli
is similar to that of a whole antibody in the eukaryotic cell. The F
v
fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant F
v
fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the F
v
fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the F
v
fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.
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