WFRF:(Gunneriusson Elin)
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- Gunneriusson, ElinKTH,Bioteknologi (author)
Display of affinity proteins on bacteria and bacteriophage
- BookEnglish1999
Publisher, publication year, extent ...
- Stockholm :Bioteknologi,1999
- 66 s.
- printrdacarrier
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- LIBRIS-ID:oai:DiVA.org:kth-2798
- ISBN:917170390X
- https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-2798URI
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- Language:English
- Summary in:English
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- SwePubSwePub
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- NR 20140805
- Surface display of peptides and proteins on microorganismshas during the last decade gained tremendous interest due tothe numerous applications for the technique found inbiotechnology, molecular biology and immunology. This thesisdescribes both bacterial display of different affinity proteinsand display of libraries of small α-helical proteindomains on phage for selection of novel binding proteinsdenoted affibodies.A single chain Fv (scFv) antibody fragment, directed againsthuman immunoglobulin E (IgE), was surface displayed on the twofood-grade staphylococcal speciesStaphylococcus xylosusandStaphylococcus carnosus, using two different expressionsystems both taking advantage of the cell wall anchoring partsof staphylococcal protein A (SpA). Heterologous,scFv-containing hybrid proteins were found to be surfaceexposed on both cell types and the whole live bacterial cellswere shown to have gained ability to bind the target antigen,human IgE.Combinatorial libraries of a 58 residue, alpha-helicaldomain derived from a SpA analogue have been monovalentlydisplayed on phage as fused to a truncated protein III of M13bacteriophages. The immunoglobulin G (IgG) Fc binding surfaceof the native domain was targeted for randomization to createlarge libraries of domain variants from which new proteins,affibodies, with novel binding specificities, could beselected. Identified affibodies directed against apolipoproteinA-1MilanoandTaqDNA polymerase, respectively, were shown toselectively bind their targets and could efficiently be used asligands in affinity chromatography to purify the targetproteins from crude lysates ofE. colicells. In addition, a high stability againstalkaline conditions was demonstrated in column sanitation stepsusing sodium hydroxide. The affinities (KD) of affibodies selected from naive libraries wereroutinely in the micromolar range, although with differentkinetic characteristics. Using a helix shuffling affinitymaturation strategy, the affinity of aTaqDNA polymerase specific affibody could be improved15-fold. Head to tail genetic fusion, yielding a divalentmolecule, was demonstrated to further increase the apparentaffinity by avidity effects.Two different affibodies, reactive with human IgA or IgE,respectively, were surface exposed onS. carnosususing the expression system employed for thescFv antibody. The affibodies were demonstrated to beproteolytically stable and efficiently and functionallydisplayed on the staphylococcal surface.Taken together, it has been demonstrated that small andhighly stable α-helical affibody domains, normally bindingto IgG, can be randomized in surface-exposed positions to yieldlarge combinatorial libraries from which new variants can beselected with redirected binding specificities. Furthermore,staphylococcal surface display of such SpA-derived affibodies,or the variable parts of an antibody linked as a scFv, has beenshown to generate recombinant bacteria with new bindingspecificities, that could for example be investigated as novelwhole cell diagnostic devices.Keywords:affibody, affinity chromatography, affinitymaturation, bacterial surface display,in vitroselection, combinatorial protein library, phagedisplay, scFv antibody fragment, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus,Zdomain.© Elin Gunneriusson,1999
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