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Science 20 May 1988:
Vol. 240. no. 4855, pp. 1038 - 1041
DOI: 10.1126/science.3285470
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Articles

Science, Vol 240, Issue 4855, 1038-1041
Copyright © 1988 by American Association for the Advancement of Science

articles

Assembly of a functional immunoglobulin Fv fragment in Escherichia coli

A Skerra and A Pluckthun

Genzentrum der Universitat Munchen, Max-Planck-Institut fur Biochemie, Martinsried, FRG.

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 Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv 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 Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv 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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