C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site
Construction of plasmids for expression of the chimeric KpSSBnStSSBc and KpSSBnPaSSBc proteins. To investigate the effect of the C-terminal domain of SSB on the size of the ssDNA-binding site, the C-terminal domain of KpSSB was replaced by that of StSSB and PaSSB. pET21b-KpSSB (Primers 7 and 8), pET21b-StSSB (Primers 9 and 10), and pET21b-PaSSB (Primers 11 and 12) vectors were mutated to create a desired SacI site and to obtain the vectors for expression of the chimeric proteins KpSSBnStSSBc and KpSSBnPaSSBc. The D91E/Q92L-engineered pET21b-KpSSB vector, the D91E/Q92L-engineered pET21b-StSSB vector, and the G90E/Q91L-engineered pET21b-PaSSB vector were cut at NdeI and SacI sites. Subsequently, the KpSSBn, StSSBc-pET21b, and PaSSBc-pET21b fragments were purified. KpSSBn was ligated with StSSBc-pET21b and PaSSBc-pET21b fragments to generate the engineered pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. To avoid artificial residues, positions 91 and 92 of the two plasmids were mutated back (Primers 13 to 16) to obtain pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc vectors. Thus, pET21b-KpSSBnStSSBc and pET21b-KpSSBnPaSSBc will express KpSSB1-91 fused StSSB92-176 and PaSSB91-165, respectively. Note that KpSSBnPaSSBc will have 166 amino acid residues.