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Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1(M) and Taq DNA polymerase
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
2000 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, no 1, 45-54 p.Article in journal (Refereed) Published
Abstract [en]

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 Tag 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.

Place, publisher, year, edition, pages
2000. Vol. 80, no 1, 45-54 p.
Keyword [en]
affibody, affinity ligand, bacterial receptor, combinatorial library, phage display, staphylococcal protein A, bacterial receptor domain, a-imilano apoprotein, plasmid dna, purification, recognition, affibody, beads
URN: urn:nbn:se:kth:diva-19832ISI: 000087626200003OAI: diva2:338524
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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