Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase.
Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Taq DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0. 5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.
Nord
K
K
Department of Biotechnology, Royal Institute of Technology (KTH), Teknikringen 30, SE-100 44, Stockholm, Sweden.
Gunneriusson
E
E
Uhlén
M
M
Nygren
P A
PA
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification; D016632:Apolipoprotein A-I / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System / Q000201:enzymology; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000378:metabolism*; D019914:Taq Polymerase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D016632:Apolipoprotein A-I; D008024:Ligands; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D019914:Taq Polymerase