Journal: The Journal of Biological Chemistry
Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
Figure Lengend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p
Article Snippet: It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells.
Techniques: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Negative Control, Western Blot