Cloning and Expression of Phosphopentomutase and Cytidylate kinase from Thermus thermophilus

Abstract

Extremophiles are unconventional microorganisms that can thrive in extreme environments. They are structurally adapted at the molecular level to withstand any harsh conditions. The biocatalysts, called extremozymes, produced by these microorganisms, are proteins that remain stable and function under extreme conditions. Due to their extreme stability, extremozymes offer new opportunities for biocatalysis and biotransformation, hence, are of great importance for industrial processes and scientific research. The extremophile that we selected is Thermus thermophilus, having an optimum growth temperature between 60°C. Bacterial phosphopentomutases (deoB) are alkaline phosphatase superfamily members that interconvert α-D-ribose-5-phosphate (ribose-5-phosphate) and α-D-ribose-1-phosphate (ribose-1-phosphate). Phosphopentomutase is a key enzyme in nucleoside catabolism. Cytidylate kinase enzyme belongs to the family of phosphotransferases. This enzyme participates in pyrimidine metabolism. Among mesophilic organisms like Streptococcus mutants, cytidylate kinase has been found to be an excellent antibiotic target. Our objective was to clone and express, selected extremophilic proteins into an expression vector. The phosphopentomutase and cytidylate kinase genes were selectively amplified. TA cloning method was selected. The amplified genes were first cloned into TA cloning (pTZ57R/T) vector and further they were subcloned into the expression vector, pET28a+. For this, vector and insert DNA were double digested with restriction enzymes and ligated with the help of T4 DNA ligase enzyme. E.coli BL21 (DE3) was used for the expression of the clones. But ligation of insert and vector was not successfully done.