关键词: 白假丝酵母；环核苷酸磷酸二酯酶；表达纯化；高效液相色谱

Abstract: For the purpose of preparing high-purity Candida albicans phosphodiesterases 2 (PDE2), the genomic DNA of C. albicans was used as a template to amplify the target gene by PCR. The expression plasmid pET28a-C. albicans pde2 was constructed, and transformed into E. coli BL21 cells to obtain genetically engineered bacteria with high expression stability. The expressed protein from the culture supernatant was consecutively purified by affinity chromatography (Ni-NAT), ion exchange column chromatography (Q-Sepharose) and Sephacryl S200 column chromatography. After that, the hydrolysis characteristics of the purified PDE2 (~62 kDa) on single and double substrates were determined by high performance liquid chromatography (HPLC). It was shown that the hydrolysis efficiency of cAMP and cGMP both at 0.4 mmol/L was 60%–70%, and the affinity was higher for cAMP. Furthermore the enzyme had high biological activity and stability. This study provides a theoretical basis for the crystallographic analysis of the protein and for understanding its role in the physiological and pathological mechanism of C. albicans in the future.

Key words: Candida albicans; cyclic nucleotide phosphodiesterase; expression and purification; high performance liquid chromatography