Cooperative effects of artificial chaperone and Mg2+ ions on alkaline phosphatase refolding

The refolding of GuHCl-denatured alkaline phosphatase (ALP) has been investigated by a new elegant strategy using the artificial-chaperone assisted two-step mechanism. However, using detergent and cyclodextrin, which are both poorly soluble in aqueous solutions, necessitates large sample dilutions to earn adequate refolding yield. In the present work, to overcome this obstacle, we studied the concomitant effects of the artificial chaperone and metal ions on ALP refolding at 0.2 mg/ml concentration. The method presented is a high-yielding process with few operational steps which may result in a low process cost. Based on this approach, refolding of GuHCl-denatured ALP raised from 20%, in the absence of anti-aggregators, to about 50% in the presence of artificial chaperone and/or MgSO4 and to 82% in the concomitant presence of the artificial chaperone and MgSO4 while the extent of aggregation was totally quenched. Using different salts of Mg2+ resulted in different recovered yields probably due to their variable interactions with cyclodextrin. Under the same experimental conditions except for the metal ions, Zn2+ and Ca2+, worked neither alone nor in combination with the artificial chaperone showing that Mg2+ can work as an auxiliary agent because of its structural role.