Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli.
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.
Liobikas
Julius
J
Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, Biomedicum Helsinki, P.O. Box 63, FIN-00014, University of Helsinki, Finland.
Polianskyte
Zydrune
Z
Speer
Oliver
O
Thompson
James
J
Alakoskela
Juha-Matti
JM
Peitsaro
Nina
N
Franck
Marina
M
Whitehead
Maria A
MA
Kinnunen
Paavo J K
PJ
Eriksson
Ove
O
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
C442861:Lactb protein, mouse; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D001618:beta-Lactamases