Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

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• 作者：Julius Liobikas ; Zydrune Polianskyte ; Oliver Speer ; James Thompson ; Juha-Matti Alakoskela ; Nina Peitsaro ; Marina Franck ; Maria A. Whitehead ; Paavo J.K. Kinnunen and Ove Eriksson
• 关键词：LACTB ; Serine protease ; β ; -Lactamase ; d-Transpeptidase ; Penicillin-binding protein ; Mitochondria
• 刊名：Protein Expression and Purification
• 出版年：2006
• 出版时间：February 2006
• 年：2006
• 卷：45
• 期：2
• 页码：335-342
• 全文大小：475 K

文摘

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the β-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione–agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His₆-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of α-helices, β-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.