Abstract:

Objective To prepare the phosphopantetheinyl transferase Ppt2 of Candida albicans, which would be used for the drug screening of Ppt2 as the target by fluorescence polarization. Methods The DNA of PPT2 was amplified by PCR using Candida albicans BWP17 genomic DNA as a template and linked with the linearized vector pET43.1a(+) by homologous recombination. The recombinant plasmid pET43.1a(+)/PPT2 was successfully constructed and transformed into E.coli BL21 (DE3) competent cells with IPTG. The protein Ppt2 was further extracted using ultrasonic fragmentation and verified solubility by SDS-PAGE. The column Ni was used to purify the protein and the elution fraction was dialyzed to enrich the target protein. The protein Ppt2 was finally determined by the Bradford method. Results The recombinant protein Ppt2 was highly expressed in E.coli BL21 with final concentration of 0.4 mg/mL. The results of SDS-PAGE electrophoresis showed that Ppt2 had both soluble form and inclusion body form. After amplification of the amount of E.Coli BL21, the soluble protein was purified by column Ni to obtain the target protein. After dialysis and enrichment of protein Ppt2, it was measured by Bradford method that protein Ppt2 concentration is 0.4 mg/mL. Conclusion The phosphopantetheinyl transferase protein Ppt2 of Candida albicans was successfully prepared, which could provide the basis for the future drug screening of Ppt2 as a target by fluorescence polarization.

Key words: Candida albicans, phosphopantetheinyl transferase Ppt2, protein preparation and purification