Inhibition of elastase and matrix metalloprotease activities accelerates the wound healing process. Human secretory leukocyte protease inhibitor (hSLPI) has been reported to demonstrate such inhibition, promoting its use as biomaterial in protein therapy. However, bioavailability of SLPI is very low and its heterologous production in Escherichia coli results in the formation of inclusion bodies, requiring refolding steps to recover its bioactivity. Current strategies to produce SLPI are hampered by either low recovery or process related limitations. Therefore, an alternative approach was developed in order to achieve soluble expression of the protein in E. coli. The gene coding for full-length human SLPI was generated from amniotic membrane through reverse-transcript PCR, and it was then cloned into the pET-101/D-TOPO vector for its expression in E. coli. Soluble recombinant hSLPI with C-terminus His tag was expressed and purified in a nickel affinity column. The purified enzyme was tested for inhibition of elastase. Soluble expression of hSLPI was achieved with the full-length protein containing its native signal peptide. Inhibition of elastase activity by the purified protein indicated that the protein is active, with an inhibition constant of ∼9 × 10-8 M, which is in similar order of magnitude to the partial hSLPI. The presence of the signal peptide appears to contribute for the soluble expression of the protein and had negligible effect on its activity.
|Number of pages||4|
|Publication status||Published - 1 Apr 2017|
- Elastase inhibition
- Escherichia coli
- Native signal peptide
- Secretory leukocyte protease inhibitor