Immunoaffinity chromatography of recombinant Amb a I in the presence of a denaturing agent.
Recombinant proteins expressed in E. coli are often sequestered into inclusion bodies and require the use of denaturing agents in order to solubilize them. The recombinant form of Amb a I, the major allergen from short ragweed pollen, is one such protein. In some cases solubility can be maintained after the removal of the denaturing agent, particularly if the protein can be folded into its native conformation. However, not all proteins refold readily and after the removal of the denaturing agent the proteins will reaggregate and/or precipitate. In the case of Amb a I, the recombinant protein stays in solution at low concentrations but aggregates with itself and other proteins. The recombinant Amb a I is not expressed at high levels and may be toxic to E. coli. Therefore, isolation from a complex mixture of E. coli proteins was necessary. Monoclonal antibodies which recognize the denatured form of Amb a I were available, allowing for immunoaffinity purification. However, because the protein was not monomeric, this chromatographic technique did not provide an improvement in the purity level when run in normal buffer solutions. Analysis of one monoclonal antibody's stability to urea indicated it could tolerate the presence of 2 M urea and recover full activity. Use of this antibody as an immunoaffinity reagent in a column run in 2 M urea, which minimized aggregation of the E. coli produced proteins, gave a high degree of purification of recombinant Amb a I in one step. This illustrates the potential for the use of denaturing and other solubilizing agents in immunoaffinity chromatography of recombinant proteins.
Keating
K M
KM
ImmuLogic Pharmaceutical Corporation, Cambridge, MA 02139.
Rogers
B L
BL
Weber
L
L
Morgenstern
J P
JP
Klapper
D G
DG
Kuo
M
M
D000485:Allergens / Q000737:chemistry / Q000302:isolation & purification*; D052179:Antigens, Plant; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D010940:Plant Proteins; D011058:Pollen; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins