Novel strategies for the purification of biomolecules by affinity chromatography: Ceramic fluorapatite binding peptides for the development of self-assembled systems and ligand-less adsorbents

  • A key issue related to bioprocessing technology is product recovery and purification i.e. the so-called downstream processing, which still remains a daunting task in the life science industry. Affinity chromatography is the most efficient method known for highly selective sequestration of (bio) products. This technique exploits the recognition ability of certain chemical structures or “ligands” for the species being targeted within a feedstock. Such ligands are normally covalently coupled to a solid support (“beads”). This traditional immobilization process, however, may present several drawbacks such as ligand inactivation and leakage, high expense, and environmental burden. To overcome such limitations, the present work has focused on the development of two completely new approaches, as follows: a) The fabrication of self-assembling affinity adsorbent beads, and b) The implementation of ligand-less affinity systems. As a first step to materialize the concepts proposed above, peptides with the ability to bind to ceramic fluorapatite beads (CFT) were discovered by screening binding-peptides out the diversity created utilizing either biological or synthetic peptide libraries. The fabrication of affinity adsorbent beads for the purification of total human immunoglobin G (hIgG) was attempted. The peptide construct pIC, composed of a hIgG-specific peptide ligand pLI, HWRGWV, and a CFT-specific peptide tag pTC, KPRSVSG (F5-4), was immobilized on CFT beads by self-assemblage without the need of any chemical procedure. The hIgG-binding capacity of the self-functionalized CFT beads increased as a function of the conjugate density on the surface of the adsorbent. The approach regarding the use of CFT-specific peptides as tags for the purification of recombinant proteins was investigated by fusing CBPs at the C terminus of enhanced green fluorescent protein (eGFP). The resulting fusion protein was expressed in Escherichia coli strain BL-21, and the clarified crude extract was subjected to CFT column chromatography. The yield and purity of the isolated protein were over 90%. This work demonstrates the potential of material binding peptides in the development of novel, and highly selective, adsorbents for chromatography and allied techniques.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Tuhidul Islam
Referee:Marcelo Fernandez Lahore, Nicolai Kuhnert, Saad Zaghloul M. ELDamrawy
Advisor:Marcelo Fernandez Lahore
Persistent Identifier (URN):urn:nbn:de:gbv:579-opus-1003058
Document Type:PhD Thesis
Date of Successful Oral Defense:2013/06/28
Year of Completion:2013
Date of First Publication:2013/09/03
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:T Technology / TP Chemical technology / TP155-156 Chemical engineering / TP156 Special processes and operations / TP156.P83 Purification
Call No:Thesis 2013/21

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