UC San Diego
Beta-Hairpin Fusion to Alpha-Helical Domains for Exploring Recombinant Protein Stability in E. coli
- Author(s): Lokensgard, Melissa E.
- Advisor(s): Love, John J
- et al.
Small α-helical domains isolated from larger multi-domain proteins, such as D. melanogaster Engrailed homeodomain (En) and Staphylococcal Protein A B domain (SpA) are recalcitrant to overexpression in BL21(DE3) E. coli hosts when using common cytosolic T7 inducible expression vectors, but they can be accumulated sufficiently for preparative research by A) fusion to a stable N-terminal domain such as Ubiquitin (Ub) or Streptococcal Protein G β1 domain (Gβ1) or B) using a vector containing the N-terminal signal sequence pelB for periplasmic localization. This dissertation describes work to show that fusion of short β-hairpin sequences derived from Ub and Gβ1 is sufficient to enhance accumulation under the same vector and host conditions. Interestingly, the short β-hairpin sequence can enhance accumulation when fused to either terminus of En or SpA. Using circular dichroism and nuclear magnetic resonance spectroscopies, we find that the fusion proteins have added β content, and that the hairpin only subtly affects thermodynamic parameters of equilibrium unfolding. Thermally destabilized variants of the two helical domains also show enhanced accumulation upon fusion to β-hairpins. Microscale thermophoresis demonstrates that the hairpins affect the extent of binding to the ClpS aptamer of the ClpAP proteolytic complex. This finding leads to the speculation that these small structural motifs can be inhibitory to E. coli protein recycling pathways active during heterologous expression, either by direct reduction of recognition or by a more complex indirect mechanism. Additionally, by adding a structurally defined unit to the ends of a domain outside its native context in a larger protein, characteristics of unstructured inter-domain regions may be able to be examined, and this technique could be useful for producing domains with unknown structure or disordered boundaries.