Bacteriophages and cryogels: A new efficient tool in bioseparation
Sammanfattning: Abstract Due to developments in the last decades in the field of protein production, new technologies for bio-separation have evolved. Especially in the field of affinity chromatography, the search for new ligands is of great interest. Besides the antibodies used as affinity ligands, synthetic ligands were introduced. Textile dyes were a first group to be introduced but despite their selectivity and capacity, the use was not very successful due to leakage and toxicity problems. More attention was focused on synthetic peptide ligands which could be synthesized using different techniques. Chemical procedures such as combinatorial chemistry, solid phase chemistry, docking simulation are applied to select peptides with affinity towards a target. By using a biological technique, Phage Display Technology, peptides can also be selected by screening peptide phage libraries towards a target. New protein production technologies have also an influence on down-stream processing techniques. Feeds such as fermentation broth, transgenic milk are particulate feed stocks which implies higher demands from the chromatographic matrices. New matrices and techniques are developed to deal with this challenge. Supermacroporous cryogel monoliths are an excellent tool for this purpose. Due to macropores with the size up to 100 µm, particulate feeds such as fermentation broth, microbial and mammalian cell cultures, milk and even whole blood can flow through the cryogel without any restriction. This thesis describes novel methods of bio-panning and as well as chromatography. Bio-panning in search for new affinity ligands is done by phage display technology. Instead of synthesizing peptides, the bacteriophages expressing a peptide on an envelope protein are used as ligands. This has several advantages compared to the pure peptide.(i) Once the phage is selected, it can be amplified in great quantities at low cost as compared to the synthesis of pure peptide ligands. (ii) By using bacteriophages a very specific ligand is available combining the effects of “affinity” and “avidity” for the optimal capture of target. By combining the cryogel monolith column with bacteriophages as affinity ligands we provided “proof of principle” for this new technique in bio-separation. Two case studies are presented. A selected peptide expressing bacteriophage clone was coupled to a cryogel monolith column. In case study one, human lactoferrin or recombinant human lactoferrin was captured from human milk or transgenic bovine milk respectively. In case study two, von Willebrand factor was captured from plasma and whole blood. In both case the captured protein of interest was recovered with a purity of >95% in a single step chromatographic procedure. Taking advantage of the structural properties of the cryogel monolith a compact bio-panning procedure, Chromato-panning, was developed. Both the screening of phage libraries as the infection of phages in E.coli cells are performed in on-column mode. As a result a significant decrease in time for bio-panning was observed. The procedure is more compact and quicker and only one panning round has to be performed in order to obtain high selective phage clones as compared to conventional bio-panning procedures.
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