PAPS is a derivative of adenosine monophosphate that is phosphorylated at the 3' position and has a sulfate group attached to the 5' phosphate. It is the most common coenzyme in sulfotransferase reactions. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Proper function of PAPSS is essential for normal physiology in human. Mutations in the PAPSS genes could cause severe disease states. In humans there are three isoforms of PAPSS: PAPSS1, PAPSS2a, and PAPSS2b which are predominantly expressed in brain, cartilage and liver respectively. PAPSS1 and PAPSS2a/b are about 73% similar in amino acid sequences. The kinetics of PAPSS formation between these isoforms are different.
We cloned PAPSS2 using pMAL-c2 and pTrc vectors. Also, we optimized expression by varying the temperature (4, 15, 20, 37 °C), time (3, 14, 20 hours and 2, 5, 7 days) and the concentration of the inducer (with 0.5 mM or 1 mM of IPTG and without IPTG). After expression, we purified protein using amylase and nickel columns. Protein was crystallized using robot also we did optimization for crystallization and tested the crystals on the diffractometer. In the future, we plan to solve structure PAPS-synthase 2 and understand the mechanism of its action.