Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification.
We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).
Blank
Kerstin
K
Biochemisches Institut, Universität Zürich, Switzerland.
Lindner
Peter
P
Diefenbach
Beate
B
Plückthun
Andreas
A
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007164:Immunosorbents; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D007128:Immunoglobulin Fragments; D007164:Immunosorbents; D011994:Recombinant Proteins