Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies

- Full length Ixolaris and its isolated Kunitz-domains were expressed in E. Coli cells.
- NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant proteins are folded.
- E. coli and High Five expressed Ixolaris exhibited similar anticoagulant activity.

Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D 1H and 2D 15N-1H NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.