Nucleotides are an essential class of biological compounds necessary for proper function, growth and replication of all living organisms. Alternative nucleotides have an application in medicine as anticancer and antiviral drugs. Newly emerging field of synthetic biology uses alternative nucleotides as the building blocks of xenonucleic acids (XNA) Phosphoribosyltransferases (PRTs) are a group of enzymes utilized by living systems to synthetise nucleotides via salvage pathway. The shared property by these enzymes is the addition of nucleobases to ribose, which is previously activated in the form of 5-phosphoribosyl 1-pyrophosphate (PRPP). The purpose of this work was to clone, express and purify five E. coli phosphoribosyltransferases and determine their optimal expression conditions, establish a purification protocol, working temperature and optimal pH as well as supstrate specificity. This work is a first step proof of the concept as part of a wider effort to produce a biosynthetic ribonucleotide synthesis pathway that would potentially allow for the production of alternative nucleotides for use in scientific research and biotechnology. Five E.coli phosphoribosyltransferases were cloned, expressed at 37°C overnight, purified by His-Tag affinity and size-exclusion chromatography. Optimal buffer for all enzymes was established to be HEPES, and pH optima for HPRT and UPRT are 7.5 and 8 respectively, while optimal temperatures are 60° and 50°C respectively; 6-mercaptopurine was found to be taken up by XPRT and HPRT but not by APRT, while 5-fluorouracil is processed by UPRT but not by OPRT