In mammals the main energy storage is represented by triacylglcerol (TAG) accumulated in lipid droplets (LD) in adipose tissue. In times of energy demand TAGs are hydrolyzed with subsequent release of non-esterified fatty acids (NEFAs) to produce energy via ?-oxidation. Lipolysis, the breakdown of TAG is a highly regulated process catalyzed by three enzymes. Adipose triglyceride lipase (ATGL) is in charge of the first and rate limiting step, as it catalyzes the release of the first NEFA from the glycerol backbone. Hormone-sensitive lipase (HSL) and the highly specific Monoacylglycerol lipase (MGL) hydrolyze the ester bonds between the second and the third fatty acid of the glycerol backbone, respectively.The minimal construct of ATGL still active in hydrolyzing TAG and localizing to the LD is 254 amino acids long and has been determined by our group (Irina Cornaciu et al, manuscript in preparation).The aim of this work was to establish a purification protocol for the minimal active fragment of mouse ATGL (mATGL254) with the objective of performing crystallization trials and biophysical experiments.Mouse ATGL 254 was cloned into several vectors and expression optimized. Purification trials were performed, where hurdles such as the occurrence of inclusion bodies and soluble aggregates have been tried to overcome. Promising results gave mATGL254 fused to Maltose-binding protein, where high yield was obtained. Although much effort has been made to prevent aggregation, the problem still needs to be solved. Smt-mATGL254 a construct that could be obtained in monomeric form but low yield was engineered to carry a TEV-protease cleavage site. Purification trials showed that exposure of the (His)6-tag is promoted under denaturing conditions only. On account of that a (His)8-tag was engineered via site-directed mutagenesis, for better binding in affinity chromatography.