Thinking beyond tradition: Polyphenols as effective refolding modulators.Int J Biol Macromol 2020IJ
Despite polyphenols having had proven roles as amyloid alleviators their service has rarely been made use of in protein refolding/renaturation thus far, where aggregation can be a major competing pathway. TGFβ3, expressed in inclusion bodies, is a classical example of a protein prone to high rate of aggregation severely limiting its refolding yield owing to its large cysteine content and structural complexity. Here, we have used various polyphenols (EGCG, baicalein, myricetin) either alone or in combination with the pseudo-chaperone beta cyclodextrin, in the refolding buffer. With the help of non-reducing SDS PAGE and size exclusion chromatography, we showed that the refolding in presence of baicalein or EGCG along with β-CD indeed increase the yield of the native protein in a time dependent manner. EGCG expedites the refolding process giving a maximum increase in the refolded protein within 24 h while baicalein takes as long as 48 h for the same. The mechanism of mode of actions of polyphenols during refolding was further delineated by ITC. The effect of polyphenols on the aggregation kinetics and stability of native TGFβ3 was also explored. Thus these small molecules provide a promising alternate route in increasing the yield of aggregation prone proteins during refolding.