ABSTRACT
M3 proteins of 44 kDa of murine gammaherpesviruses 68 and 72 (MHV-68, MHV-72) were identified as herpesvirus vCKBP-3, soluble inhibitors of the host chemokine network providing a selective advantage for the virus by inhibiting the antiviral and inflammatory response during both acute and latent infection. The MHV-72 M3 protein was found to contain a single mutation (Asp307Gly) near its chemokine-binding domain and differ from that of MHV-68 in the ability to bind some human chemokines. In this study, we optimized the expression of his-tagged M3 proteins of MHV-68 and MHV-72 in Escherichia coli and their purification by Ni-NTA chromatography under both denaturing and native conditions. The integrity of the N-terminus of the MHV-72 M3 protein was verified by partial sequencing. The results showed that E. coli is useful for the preparation of native, recombinant M3 proteins of murine gammaherpesviruses in sufficient quantity and purity for further biological studies.
Subject(s)
Herpesviridae Infections/virology , Rhadinovirus/metabolism , Viral Proteins/isolation & purification , Animals , Blotting, Western , Cell Line , Chromatography, Ion Exchange , Cricetinae , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Gene Expression , Genetic Vectors , Humans , Mice , Mutation , Nitrilotriacetic Acid/analogs & derivatives , Phosphatidylethanolamines , Recombinant Proteins , Rhadinovirus/genetics , Sequence Analysis, Protein , Species Specificity , Transgenes , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and purified to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3(1)21 and tetragonal P4(1)2(1)2, with unit-cell parameters a = b = 64.7, c = 69.6 A, gamma = 120 degrees and a = b = 34.0, c = 147.2 A, respectively. They diffract to 2.0 and to 1.7 A resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V(M) = 3.8 A(3) Da(-1) with a solvent content of 68% and V(M) = 1.9 A(3) Da(-1) with a solvent content of 37%, respectively.
