Identification of Ss-LrpB specific Nanobodies and their application in chromatin immunoprecipitation (ChIP)

Student thesis: Master's Thesis

Abstract

Chromatin Immunoprecipitation (ChIP) is considered as a critical step in current standard methods for the analysis of in vivo transcriptional regulation mechanisms. The most important factor in ChIP is the antibody which offers the ability to specifically capture certain protein-DNA complexes. The source of antibody for ChIP assay can be polyclonal or monoclonal. The drawbacks with polyclonal antibodies are that their sources are limited, and there is batch to batch variability so that for each batch the conditions should be optimized. Moreover, the polyclonal antibodies exhibit a high non-specific background. Monoclonal antibodies do not suffer from the above-mentioned problems but not all are suitable to capture antigen from solution.
Nanobodies or VHHs are single domain antibodies derived from camel Heavy-chain only antibodies (HCAbs). Nanobodies can be obtained as monoclonal antibodies by cloning the variable regions of the HCAbs. Nanobodies are easy and cheap to produce in commonly-used expression hosts such as E. coli, and bind their targets with hgih affinity and specificity. In this study, we aim to address the possibility to use Nanobodies for ChIP. As a model transcription regulator we used Ss-LrpB, a well-known transcriptional regulator of Sulfolobus solfataricus, a hyperthermophilic archaeon.
To this end, we first generated and identified Nanobodies specific for Ss-LrpB by immunization in an alpaca with Ss-LrpB, and construction and screening of a VHH (Nanobody)phage display library from this animal. We then expressed and purified the Ss-LrpN-specific Nanobodies and characterized them in terms of overlap between epitopes and affinity. Finally, using some of these Ss-LrpB-specific Nanobodies, we demonstrated that Nanobodies can be considered as a new class of reagents for affinity capture of protein-DNA complexes in ChIP. Taking into account the advantages of Nanobodies over other antibody formats such as Fab, scFv and, VH, our results open the gate to develop novel Nanobody-based ChIp asays. Moreover, our results provide a basis to further unravel the target genes regulated by Ss-LrpB.
Date of Award1 Sep 2008
Original languageEnglish
SupervisorSerge Muyldermans (Promotor), Gholamreza Hassanzadeh Ghassabeh (Co-promotor) & Eveline Peeters (Co-promotor)

Keywords

  • Ss-LrpB specific Nanobodies
  • chromatin immunoprecipitation

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