Influence of protein solution in nucleation and optimized formulation for the growth of ARM lipase crystal

ARM lipase is a thermostable and organic solvent tolerant enzyme which was highly purified prior to crystallization. The His-tagged ARM lipase was purified with immobilized metal affinity chromatography followed by anion-exchange chromatography. The effect of different salt concentrations on stability, solubility and crystal nucleation of the protein was studied. The highly purified and homogeneous ARM lipase with protein concentration of 2 mg/mL was successfully crystallized by a sitting drop, vapor diffusion method with the use of 0.1 M MES monohydrate pH 6.5 and 12% (v/v) polyethylene glycol (PEG) 20000 as precipitant. The crystallization conditions were optimized by changing the pH and concentration of the precipitant. The optimum crystallization condition was 2 mg/mL ARM lipase in 0.1 M Tris-HCl, 0.15 M NaCl, pH 8.0 protein solution, crystallized using 0.1 M Tris-HCl, pH 8.0 and 12% (v/v) PEG 20000 as precipitant.