Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization

• rCmPI-II and 15N rCmPI-II was obtained in Pichia pastoris expression system.
• rCmPI-II showed similar functional and molecular characteristics to the natural inhibitor.
• 15N rCmPI-II inhibits trypsin and Subtilisin A with same strength to rCmPI-II.
• NMR spectra confirmed the correct folding of recombinant inhibitors.
• HSCQ and 3D NMR spectra allowed a partially CmPI-II backbone assignment.

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or 15N-labeled forms using ammonium chloride (15N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and 15N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.