Engineered α-hemolysin pores with chemically and genetically-fused functional proteins

Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane  barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.EThOS - Electronic Theses Online ServiceGBUnited Kingdo