title: Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli.
abstract: LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-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 alpha-helices, beta-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.
authors.affiliation: Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, Biomedicum Helsinki, P.O. Box 63, FIN-00014, University of Helsinki, Finland.
authors.forename: Julius
authors.initials: J
authors.lastname: Liobikas
authors.forename: Zydrune
authors.initials: Z
authors.lastname: Polianskyte
authors.forename: Oliver
authors.initials: O
authors.lastname: Speer
authors.forename: James
authors.initials: J
authors.lastname: Thompson
authors.forename: Juha-Matti
authors.initials: JM
authors.lastname: Alakoskela
authors.forename: Nina
authors.initials: N
authors.lastname: Peitsaro
authors.forename: Marina
authors.initials: M
authors.lastname: Franck
authors.forename: Maria A
authors.initials: MA
authors.lastname: Whitehead
authors.forename: Paavo J K
authors.initials: PJ
authors.lastname: Kinnunen
authors.forename: Ove
authors.initials: O
authors.lastname: Eriksson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: C442861:Lactb protein, mouse; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D001618:beta-Lactamases