The　trans-translation　system　is　recognized　as　an　excellent　target　for　developing　new　drugs　to　rapidly　sterilize　Mycobacterium　tuberculosis　(TB)　infection　and　significantly　shorten　TB　treatment　duration.　As　a　vital　component　of　the　trans-translation　system　for　rescuing　stalled　ribosomes,　the　SmpB　protein　from　Mycobacterium　tuberculosis　(MtbSmpB,　1-160　a.　a.)　mediates　tmRNA　binding　to　stalled　ribosomes　through　forming　a　complex　with　tmRNA.　So　far,　few　works　have　been　conducted　to　prepare,　characterize　biophysical　properties　and　determine　three-dimensional　structure　for　the　full-length　MtbSmpB　protein.　In　the　present　work,　we　successfully　expressed　and　purified　the　His-tagged　full-length　MtbSmpB　protein　in　Escherichia　coli　with　a　yield　of　26.9　mg　from　1　L　of　Luria　Bertani　medium.　We　also　obtained　MtbSmpB　with　a　yield　of　18.5　mg　from　1　L　of　M9　minimal　medium.　The　MtbSmpB　protein　showed　a　single　band　in　SDS-PAGE　with　a　molecular　weight　of　similar　to　20　kDa　consistent　with　the　measurement　from　MALDI-TOF-mass　spectrometry.　The　dynamic　light　scattering　experiment　indicated　that　MtbSmpB　existed　in　a　monomeric　form.　Moreover,　both　circular　dichroism　and　nuclear　magnetic　resonance　(NMR)　experiments　exhibited　that　MtbSmpB　was　well　structured,　suggesting　that　it　could　be　feasible　to　determine　its　solution　structure　by　NMR　spectroscopy.　NMR　titration　experiments　showed　that　MtbSmpB　specifically　bound　to　tmRNA.　This　work　lays　the　essential　basis　for　further　determining　the　solution　structure　and　dynamics　of　the　full-length　MtbSmpB　protein.