Nanobodies (Nbs) are the small (15 kDa) and stable single domain fragments derived from variable region of heavy chain antibodies produced by camelids, which harboring the full antigen-binding capacity of original antibodies. The high stability and easy production in various micro-organism makes nanobodies convenient tools for numerous bioanalytical and biotechnical applications. Recently was reported a nanobody reffered to as BC2 (nanobody against beta catenin) which recognizes a short linear epitope (PDRKAAVSHWQQ, reffered to as BC2 tag) with high affinity (KD ~1.4 nM) and selectivity [1,2]. This type of binding interaction was used in our work for preparing the protocol for purification and detection of selected RNA dependent RNA polymerases (RdRp). We cloned BC2 Nb and recombinant RdRp with BC2 tag on C-terminus into the pRSFDuet vector and pSUMO vector, respectively, and expressed in E. coli BL21-CodonPlus-RIL cells. Firstly, we purified both using classical IMAC techniques and after that we tested binding affinity and influence of BC2 tag on RdRp. We were fighting with misfolding of BC2 Nb, but at the end we solved the problem using denaturating buffer with urea and refolding of nanobody. Finally, we established purification protocol of BC2 Nb and confirmed the activity of BC2 tagged RdRp alone and also in complex. Our next aim is use BC2 Nb as main purification system for RdRp, because purification by IMAC in many cases leads to the precipitation of this type of proteins.
The work was supported from European Regional Development Fund; OP RDE; Project: "Chemical biology for drugging undruggable targets (ChemBioDrug)" (No. CZ.02.1.01/0.0/0.0/16_019/0000729).