Heterologous overexpression of genes in Escherichia coli has made it possible to obtain high titers of recombinant proteins. However, this can result in the formation of aggregated protein particles known as inclusion bodies'. Protein sequestered as inclusion body is inactive and needs to be converted back to its functional form by refolding using appropriate techniques. In the current study inclusion bodies of the enzyme aminoglycoside nucleotidyl transferase (or ANT(2)-Ia) were first solubilized in urea and subsequently subjected to thermal cycling under controlled conditions as part of the refolding strategy. Thermal cycling led to disaggregation of the individual protein chains and simultaneously refolding the released protein molecules to their native state. The optimum condition was identified as 10-80 degrees C thermal cycling at 3 degrees C s(-1) for 2 h. Enzyme activity measurements showed that thermal cycling under optimized conditions resulted in 257% activity recovery when compared with nonrefolded protein. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:133-139, 2017