Expression and Purification of the Cytoplasmic Domain of BlaR1; poster

FISHOVITZ, J.; LLARRULL, L.I.; LASTOCHKIN, E.; MOBASHERY S.

Plymouth, Indiana

Jornada; 16th ANNUAL BIOCHEMISTRY RESEARCH FORUM; 2011

Department of Chemistry & Biochemistry, University of Notre Dame

The BlaR1 protein has been implicated in the mechanism of beta-lactam resistance of Staphylococcus aureus. It is a membrane protein that includes four transmembrane segments(TM1-4) connected by loops (L1-L3). The L3 loop is located in the cytoplasm and is described as a metalloprotease. Upon activation, this domain of BlaR1 is responsible for the degradation and subsequent removal of the BlaI repressor of the bla operon, leading to transcription of the blaZ gene and production of beta-lactamase, culminating in antibiotic resistance. Currently, the L3 loop has yet to be isolated as a pure, soluble protein. In an effort to recover a significant amount of soluble L3 protein for crystallographic studies, we have designed two constructs of this cytoplasmic loop (cytBlaR), with the addition of a solubility tag to aid in expression and purification. A combination of affinity and size-exclusion chromatography is used to purify cytBlaR from the protein tag. In the present study, we use Western Blot analysis monitor the degradation  of purified BlaI as a measure of activity of the purified cytBlaR protein. Beta-lactam resistance of Staphylococcus aureus. It is a membrane protein that includes four transmembrane segments(TM1-4) connected by loops (L1-L3). The L3 loop is located in the cytoplasm and is described as a metalloprotease. Upon activation, this domain of BlaR1 is responsible for the degradation and subsequent removal of the BlaI repressor of the bla operon, leading to transcription of the blaZ gene and production of beta-lactamase, culminating in antibiotic resistance. Currently, the L3 loop has yet to be isolated as a pure, soluble protein. In an effort to recover a significant amount of soluble L3 protein for crystallographic studies, we have designed two constructs of this cytoplasmic loop (cytBlaR), with the addition of a solubility tag to aid in expression and purification. A combination of affinity and size-exclusion chromatography is used to purify cytBlaR from the protein tag. In the present study, we use Western Blot analysis monitor the degradation  of purified BlaI as a measure of activity of the purified cytBlaR protein.