Mycobacterium　tuberculosis　(Mtb)　is　the　key　devastating　bacterial　pathogen　responsible　for　tuberculosis.　Increasing　emergence　of　multi-drug-resistant,　extensively　drug-resistant,　and　rifampicin/isoniazid-resistant　strains　of　Mtb　makes　the　discovery　of　validated　drug　targets　an　urgent　priority.　As　a　vital　translational　component　of　the　protein　biosynthesis　system,　elongation　factor　Tu　(EF-Tu)　is　an　important　molecular　switch　responsible　for　selection　and　binding　of　the　cognate　aminoacyl-tRNA　to　the　acceptor　site　on　the　ribosome.　In　addition,　EF-Tu　from　Mtb　(MtbEF-Tu)　is　involved　in　the　initial　step　of　trans-translation　which　is　an　effective　system　for　rescuing　the　stalled　ribosomes　from　non-stop　translation　complexes　under　stress　conditions.　Given　its　crucial　role　in　protein　biosynthesis,　EF-Tu　is　identified　as　an　excellent　molecular　target　for　drug　design.　Here,　we　reported　the　recombinant　expression,　purification,　biophysical　characterization,　and　structural　modeling　of　the　MtbEF-Tu　protein.　Our　results　demonstrated　that　prokaryotic　expression　plasmids　of　pET28a-MtbEF-Tu　could　be　expressed　efficiently　in　Escherichia　coli.　We　successfully　purified　the　6x　His-tagged　proteins　with　a　yield　of　16.8　mg　from　1　l　of　Luria　Bertani　medium.　Dynamic　light　scattering　experiments　showed　that　MtbEF-Tu　existed　in　a　monomeric　form,　and　circular　dichroism　experiments　indicated　that　MtbEF-Tu　was　well　structured.　Moreover,　isothermal　titration　calorimetry　experiments　displayed　that　the　purified　MtbEF-Tu　protein　possessed　intermediate　binding　affinities　for　guanosine-5-triphosphate　(GTP)　and　GDP.　The　GTP/GDP-binding　sites　were　predicted　by　flexible　molecular　docking　approach　which　reveals　that　GTP/GDP　binds　to　MtbEF-Tu　mainly　through　hydrogen　bonds.　Our　work　lays　the　essential　basis　for　further　structural　and　functional　studies　of　MtbEF-Tu　as　well　as　MtbEF-Tu-related　novel　drug　developments.