کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020195 1542315 2016 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization
چکیده انگلیسی


• 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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 126, October 2016, Pages 127–136
نویسندگان
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