Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli.

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.