Figure 1.
Expression, purification and crystallization of BnDGAT1(1–116)His6. Panel A: Induction of BnDGAT1(1–116)His6 expression in bacteria monitored by SDS-PAGE. From left to right: molecular mass markers (lane 1), clarified lysate from non-induced bacteria (lane 2), clarified lysate from two independent incubations of bacteria treated with IPTG (lanes 3 and 4), pellet obtained from clarified lysate of bacteria induced with IPTG (lane 5). Experiments to demonstrate induction of BnDGAT1(1–116)His6 synthesis were conducted using an incubation medium of 5 ml (instead of the 500 mL used for preparative work). Harvested E. coli were resuspended in 100 mM phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French pressure cell at 20,000 p.s.i. three times and the ruptured bacteria were centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The 13,000 g pellet obtained from ruptured bacteria that had been induced with IPTG was resuspended in extraction buffer. Equal volumes of each sample were applied to the SDS gel. Panel B: Analysis by SDS-PAGE of BnDGAT1(1–116)His6 purified by immobilized nickel ion chromatography. From left to right: molecular mass markers (lane 1), pass-through fraction (lane 2), fraction eluted with equilibration buffer (lane 3), and fractions eluted with 4 ml portions of equilibration buffer containing 40, 60 and 80 mM imidazole, respectively (lanes 4–6). The protein load for lane 4 was about 10 μg. Ten microliter aliquots of each of the eluents were applied to the gel. Panel C: MALDI-TOF spectrum of BnDGAT1(1–116)His6. The spectrum was acquired using a Voyager DE-Pro TOF MS (Applied Biosystems, Foster, CA, USA) using a 2-layer spotting technique. Matrix layer 1 was approximately 15 mg/mL α-cyano-4-hydroxycinnamic acid dissolved in 1:2 (v/v) methanol:acetone. Matrix layer 2 was saturated α-cyano-4-hydroxycinnamic acid in 1:2 (v/v) methanol/water. One microliter of layer 1 solution was spotted and allowed to dry. Protein sample (450 μg/mL) was mixed with layer 2 solution in a ratio between 1:1 and 1:4 (sample:matrix). One microliter of layer 2 solution was spotted on the dried layer 1 spot, and allowed to dry. The dried spot was washed 2–3 times with a drop of cold distilled water to remove salts prior to analysis by MALDI-TOF mass spectrometry. Panel D: Microcrystals of BnDGAT1(1–116)His6. To begin preparation of microcrystals, a 12 mg/mL solution of BnDGAT1(1–116)His6 was dialyzed against 10 mM glycine-NaOH (pH 9.0) containing 20 mM NaCl and 1 mM EDTA. Initial screening for crystallization conditions using vapor diffusion and hanging drops resulted in several instances of microcrystals, which grew as plates, within 1–2 weeks. Microcrystals were obtained from ammonium sulfate and ammonium phosphate solutions over a wide pH range.