Auto-induction and purification of a Bacillus subtilis transglutaminase (Tgl) and its preliminary crystallographic characterization.
Spores of Bacillus subtilis are covered by a multi-protein protective coat which is a key factor in their extreme environmental resilience. A fraction of the coat proteins undergoes covalent cross-linking following their assembly at the spore surface. Several types of covalent cross-links are found in the coat. These include epsilon-(gamma-glutamyl)lysine bonds whose formation is catalyzed by a transglutaminase, Tgl, itself a coat component. Tgl is the smallest known transglutaminase. It bears no sequence resemblance to other proteins in databases, except for its counterparts in other Bacillus and related species, suggesting a highly specialized role in coat assembly. It is not known to what degree are the Tgl-like proteins structural and mechanistically related to other transglutaminases. Here, we have fused the His(6) tag to the C-terminal end of Tgl, and shown that the fusion protein is functional in vivo. We have overproduced B. subtilis Tgl-His(6) by auto-induction with high yield and purified the protein to nearly homogeneity in a single chromatographic step. The purified protein, active as it catalyzed the cross-linking of bovine serum albumin, behaved as a monomer of about 33kDa in solution. Lastly, Tgl was crystallized and X-ray diffraction data were collected using synchrotron radiation to 2.1A resolution. Crystals of Tgl belong to the tetragonal space group P4(1,3) and contain two molecules per asymmetric unit.
Plácido
Diana
D
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, ITQB-UNL, Av. República, Apt. 127, 2781-901 Oeiras, Portugal.
Fernandes
Catarina G
CG
Isidro
Anabela
A
Carrondo
Maria Arménia
MA
Henriques
Adriano O
AO
Archer
Margarida
M
D001412:Bacillus subtilis / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D013171:Spores, Bacterial / Q000201:enzymology; D011503:Transglutaminases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D011503:Transglutaminases