Single-step recovery of a secreted recombinant protein by expanded bed adsorption.
We have used an expanded bed adsorption procedure for efficient recovery of a recombinant fusion protein, directly from a crude fermentor broth without prior cell removal. The fusion protein was designed to have a relatively low isoelectric point (pI) to allow anionic exchange adsorption at pH 5.5 where most Escherichia coli host proteins are not adsorbed. The gene product was secreted to the culture medium of the E. coli host cells in high yields (550 mg/l). The separation of cells and the concentration and recovery of the fusion protein could therefore be achieved by a single unit operation. The yield after the expanded bed adsorption exceeded 90 percent. Furthermore, the significant volume reduction by the expanded bed adsorption, enabled efficient and straight-forward polishing of the product by a subsequent affinity chromatography step, for removal of contaminating DNA and pyrogenic compounds to levels acceptable for regulatory authorities. An overall yield exceeding 90 percent was maintained after the affinity chromatography polishing step. The procedure outlined here is suitable for large-scale bioprocesses and allows efficient removal of cells, host proteins, contaminating DNA and endotoxins.
Hansson
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Ståhl
S
S
Hjorth
R
R
Uhlén
M
M
Moks
T
T
D000327:Adsorption; D000818:Animals; D000838:Anions; D000953:Antigens, Protozoan / Q000235:genetics / Q000302:isolation & purification; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics / Q000302:isolation & purification
D000838:Anions; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C050256:ring-infected erythrocyte surface antigen (RESA), Plasmodium falciparum