LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in Delta T = 10 degrees C increments within the range T = 55-95 degrees C; and the strongest affinity binding was disrupted at T = 85 degrees C where C-Apt = 0.16 mu M was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K-D = 218 nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein. (C) 2009 Elsevier Inc. All rights reserved.