Competitive elution of protein A fusion proteins allows specific recovery under mild conditions.
A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K(aff)) to polyclonal human IgG was found to be 3.3 (+/- 0.4) x 10(8) M-1 for divalent ZZ and 2.0 (+/- 0.1) x 10(7) M-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.
Nilsson
J
J
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Nilsson
P
P
Williams
Y
Y
Pettersson
L
L
Uhlén
M
M
Nygren
P A
PA
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004256:DNA Polymerase I / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D004274:DNA, Recombinant; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004256:DNA Polymerase I