Johan Nakuci

University of Rochester

Drennan Laboratory
Mentor: Yan Kung

Summer 2007

Crystalization of Epothilone Protein Subunit EpoB-Cy

Epothilones are nonribosomal-peptide-polyketidenatural products constructed by an assembly line of nonribosomal-peptide synthetases (NRPS) and polyketide synthases (PKS) involving proteins EpoA-F.   Our interest is in the EpoB-Cy subunit of the NRPS assembly line. EpoB-Cy gene was amplified using polymerase chain reaction (PCR) from the existing constructs or from genomic DNA and ligated into pET28 a . Construct amplification was conducted by transforming pET28 a -EpoB-Cy into XL1-Blue E. coli competent cells with kanamycin as the selection marker. Presence of the construct in the competent cells was further tested via a restriction digest with XhoI and NdeI. EpoB-Cy subunit was expressed in BL21(DE3)Rossetta2 E. coli cells under the control of a T7 promoter at 37 ° C and expression was induced using IPTG.   Preliminary protein gels, used to check the concentration of the protein expressed, revealed not a significant amount of EpoB-Cy was expressed. This failure is believed to be the result of not incubating the cells long enough while protein expression was induced. Once this problem is circumvented the protein will be purified by nickel chromatography and crystallization plates will be set up.