Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion.
The predicted ribonuclease (RNase) HI domain of the open reading frame Rv2228c from Mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (MBP) fusion. Expression was only observed for the MBP-fusion protein, which was purified using amylose affinity chromatography and gel filtration. The RNase HI domain could be cleaved from the MBP-fusion protein by factor Xa digestion, but was very unstable. In contrast, the fusion protein was stable, could be obtained in high yield and gave crystals which diffracted to 2.25 A resolution. The crystals belong to space group P2(1) and have unit-cell parameters a = 73.63, b = 101.38, c = 76.09 A, beta = 109.0 degrees. Two fusion-protein molecules of 57,417 Da were present in each asymmetric unit.
Watkins
Harriet A
HA
Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, 3A Symonds Street, Private Bag 92019, Auckland, New Zealand. h.watkins@auckland.ac.nz
Baker
Edward N
EN
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D058619:Maltose-Binding Proteins; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D017931:DNA Primers; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D016914:Ribonuclease H; C078856:ribonuclease HI