Protein Expression and Purification 45 (2006) 335–342 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.08.006 Expression and puriWcation of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli Julius Liobikas a,1 , Zydrune Polianskyte a , Oliver Speer a , James Thompson b , Juha-Matti Alakoskela a , Nina Peitsaro a , Marina Franck a , Maria A. Whitehead b , Paavo J.K. Kinnunen a , Ove Eriksson a,¤ a Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, Biomedicum Helsinki, P.O. Box 63, FIN-00014, University of Helsinki, Finland b Androgen Receptor Laboratory, Institute of Biomedicine/Physiology, Biomedicum Helsinki, P.O. Box 63, FIN-00014, University of Helsinki, Finland Received 13 May 2005, and in revised form 6 August 2005 Available online 13 September 2005 Abstract LACTB is a mammalian mitochondrial protein sharing sequence similarity to the -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 aYnity 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 His 6 -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 -helices, -sheets and turns, consistent with a well-deWned 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.  2005 Elsevier Inc. All rights reserved. Keywords: LACTB; Serine protease; -Lactamase; D-Transpeptidase; Penicillin-binding protein; Mitochondria LACTB is a 60 kDa mammalian protein of unknown function sharing signiWcant sequence similarity to -lacta- mases and penicillin binding proteins (PBPs) 2 occurring in bacteria [1]. -Lactamases and PBPs are involved in the synthesis of the peptidoglycan layer in bacteria. Mouse LACTB is 551 amino acids long and comprises of a pre- dicted mitochondrial import sequence, a short putative transmembrane segment, and a -lactamase homology domain containing the serine protease motif, –SXXK– [2]. Since peptidoglycan is not synthesized by eukaryotes the role of LACTB in mammalian cells is intriguing. LACTB has been detected in mammalian heart and liver mitochondria [3,4]. Furthermore, LACTB is associated with the 39S subunit of the mammalian mitochondrial ribosome [5]. Interestingly, LACTB and Wve other addi- tional proteins of the 39S subunit of the mammalian mitochondrial ribosome lack homologs in the bacterial ribosome and yeast mitochondrial ribosome [5]. Therefore, these uniquely mammalian mitochondrial ribosomal proteins may have other additional functions unrelated to mitochondrial protein synthesis [6]. The ability of * Corresponding author. Fax: +358 9 191 25 444. E-mail address: ove.eriksson@helsinki.W (O. Eriksson). 1 Present address: Laboratory of Biochemistry, Institute for Biomedical Research, Kaunas University of Medicine, Eiveniu 4, LT-50009 Kaunas, Lithuania. 2 Abbreviations used: PBP, penicillin-binding protein; mLACTB, mouse LACTB; MALDI-TOF, matrix-assisted laser-induced ionisation/desorp- tion and time-of-Xight mass spectrometry; FTIR, fourier transform infra- red spectrometry.