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Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-5391-600X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
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2003 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 16, no 12, 1147-1152 p.Article in journal (Refereed) Published
Abstract [en]

Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD* dimer. The ABD* dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution ( pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.

Place, publisher, year, edition, pages
2003. Vol. 16, no 12, 1147-1152 p.
Keyword [en]
affinity chromatography, capacity, protein G, purification, serum albumin-binding domain, human-serum-albumin, mutagenesis, stability, peptides
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-23282DOI: 10.1093/protein/gzg121ISI: 000220520400036OAI: diva2:341980
QC 20100525 NR 20140804Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2012-02-14Bibliographically approved

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Gräslund, TorbjörnNygren, Per-ÅkeHober, Sophia
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