Expression and purification of Xcm(I) endonucleases from xanthomonas campestris in E. coli using pET expression system.

Restriction endonucleases are enzymes that exist in restriction modification (RM) systems that occur mainly among prokaryotes this system serves as a defensive system protecting host against foreign DNA invasion. Four type of restriction enzymes have been found and classified based on their subunit composition, cofactor necessity and mode of function. Restriction enzymes have prominent importance for gene analysis and cloning work in biotechnological research. Type (II)P restriction enzymes have a remarkable role in genetic engineering because of their ability to recognize a specific palindromic site and cleavage the same site. Many restriction enzymes extracted from different bacteria strains. Xcm (I) is a type (II) P restriction enzyme that extracted from xanthomonas campestris. In this study The gene encoding Xcm (I) was PCR- -amplified from the genomic DNA of xanthomonas campestris as the template, using the Pfu DNA polymerase (Fermentas, Germany) and designed specific primers. The PCR amplicon was digested with Xho1 and Nde1 and ligated into the pET 26 (b+) vector and transformed into E. coli DH5α strain. The resulting recombinant plasmid was then transformed into E. coli BL21 (DE3) for expression. The cloning process was verified by double digestion and sequencing analysis. The recombinant Xcm (I) was expressed in optimum culture condition. The tagged enzyme was prepared in pET vector and purified using Ni-NTA beads. The expressed and purified Xcm (I) enzyme was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using negative and positive controls.

Expression, Xcm, endonucleases