Figure 2
Solubilization and formation of heat-induced aggregates (HIAs) of TkAMPpase. (a) SDS-PAGE analysis of 4 µL both soluble (S) and insoluble (IS) protein fractions of OD600 = 5 samples after the expression of TkAMPpase in E. coli using Enpresso B medium. The TkAMPpase (54 kDa) is mainly detected in the insoluble fraction (IS) after cell disruption using BugBuster Protein Extraction Reagent. M: protein marker. (b) 5 µL of the protein fractions obtained from French Press cell disruption at 900 bar analyzed by SDS-PAGE. (c) The fifth French Press fraction of the soluble protein was further purified by heat treatment at 80 °C for 30 min which resulted in a significant re-aggregation of the protein and its accumulation in the insoluble fraction. The marker (M) lane and the sample lanes are rearranged from the same gel. The complete SDS-PAGE from (a), (b) and (c) are shown in the supplementary material. (d) The induction of aggregation by heat at different temperatures within 1 h. Room temperature (approximately 25 °C) was used as a negative control to examine spontaneous aggregation. Chart is generated based on densitometric analysis of SDS–polyacrylamide gels using ImageJ software. (e) The specific activity of all aggregates was determined with 2 mM CMP and 50 mM phosphate in 50 mM MOPS buffer (pH 7.5) at 80 °C.
