The shorter version of the fusion protein, Rap1– Rif236, was created from pHK35 using the mutagenesis method described above for the generation of single amino acid changes removing codons 37 60 (HK121, HK123) to create pHK68.
This construct was then used as template for the in situ mutagenesis reaction using the primers described above.
Site-directed mutagenesis was carried out by using the methods described by Horton or the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands).
Mutagenesis reactions were performed using the method described by Kunkel (14) using 5-HT3A receptor subunit cDNA (Accession: AY605711) in pcDNA3.1 (Invitrogen, Paisley, UK) as described previously (15).
Mutagenesis was carried out on the pUB26 nuhm) plasmid using the protocol described above for the pMW172 constructs (see Supplementary Table S2).
TBCC constructs were assembled using point mutagenesis and isothermal assembly, expressed in bacteria, and purified using the approach described above with few modifications.
Recombinant TTR (rTTR) was expressed and purified using the method described by Kingsbury et al. Modification of the expression vector for rTTR variant synthesis was achieved via site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
Mutagenesis of alsSD and nuc was done as previously described [52] using the pKOR1 mutagenesis system [45].
The mutant RAP1 – RIF8Y60 F8andF8W, and fusionsion genes were created using the site-directed mutagenesis method described above for the generation of single amino acid changes using pHK35 as a template.
Preparation of mutagenic constructs, restriction digestion, ligation and transformation were performed using the PCR ligation mutagenesis strategy (insertion deletion mutagenesis) previously described [ 22].
