Open AccessCloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose‐binding protein fusion
Author(s) -
Watkins Harriet A.,
Baker Edward N.
Publication year - 2008
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309108021118
Subject(s) - maltose binding protein , fusion protein , rnase p , affinity chromatography , chemistry , mycobacterium tuberculosis , protein a/g , biochemistry , microbiology and biotechnology , biology , recombinant dna , rna , enzyme , gene , tuberculosis , medicine , pathology
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 Å resolution. The crystals belong to space group P 2 1 and have unit‐cell parameters a = 73.63, b = 101.38, c = 76.09 Å, β = 109.0°. Two fusion‐protein molecules of 57 417 Da were present in each asymmetric unit.