Fig. 6.

BrLTP2.1 is extremely heat stable. (A) One milliliter of E. coli cell culture was harvested 4 h after induction of BrLTP2.1 expression with IPTG and directly boiled for the indicated times. Boiled samples were incubated on ice for 15 min and centrifuged at 17000 g for 10 min to precipitate denatured protein and cell debris. Remaining proteins were evaluated by SDS-PAGE. (B) SDS-PAGE analysis of the clarified supernatant of cell cultures boiled for 15 min (left lane) and further purified BrLTP2.1 via Co²⁺ affinity chromatography (right lane). (C) Western blot analysis of boiled and purified BrLTP2.1 from (B) as detected by anti-His-tag antibodies. Arrowheads indicate the multiple bands corresponding to BrLTP2.1 post-boil and affinity purification. (D) Boiled BrLTP2.1 retains lipid-binding activity after purification [from (B)], as determined by displacement of TNS with palmitic acid.