Tuhidul Islam

• 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.