Ten-milliliter aliquots of induced protein-expressing cells are harvested (2060g, 10 min at 4°) and resuspended in 1.5 ml 50 mM phosphate buffer, pH 8.0, containing 300 mM NaCl. When samples are analyzed over the time course of expression, the cell concentration should be adjusted to the same value by dilution with fresh medium. We usually use OD values at 600 nm as arbitrary units for the cell number. The cells are lysed by sonication (3 min, 30% duty cycle) after treatment with lysozyme (500 mg/ ml, 30 min on ice) and DNase (50 mg/ml, 15 min on ice). The cell homog-enate is separated into soluble and insoluble fractions by centrifugation at 27,000g for 30 min. The insoluble pellet is solubilized in 1.5 ml of the same buffer containing 8 Murea. Both fractions are analyzed on SDS-PAGE, and bands are quantified for the protein of interest in each fraction by optical densitometry (Bio-Rad).
For tighter control of the uptake of osmolytes (proline, glycine betaine), we used a ProP deletion strain (E. coli WG 710) with an exogenous copy of the ProP autotransporter on the pDC80 (AmpR) plasmid under the control of the arabinose promoter (Racher et al., 2001). Both tetra-Cys CRABP and P39A tetra-Cys CRABP were subcloned in a pHSG398 vector (Takara Bio, Tokyo, Japan) plasmid bearing chloramphenicol resistance and under the control of the Ptac promotor. The ProP transporter can be induced with 0.2% arabinose and activated with 300 mM NaCl concomitantly with induction of the biosynthesis of the tetra-Cys CRABP proteins at OD600 = 1.0. Pulse induction (0.2% arabinose) and activation (300 mM NaCl) of the ProP transporter 10 min before the induction of the protein with 0.4 mM IPTG and subsequent change to a fresh MOPS medium (4 g/liter glycerol, 40 mM MOPS, 4 mM tricine, 15 mM KH2PO4, 1.5 mM K2HPO4, 15 mM NaCl, 9.5 mM NH4Cl, 0.5 mM MgSO4, 0.2 mM FeSO4, 50 mM CaCl2, pH 7.4; additionally supplied with 300 mM NaCl) yielded the same results.
Exogenous osmoprotectants, readily available in the nutrient medium, have priority over the metabolically costly biosynthesis of endogenous osmolytes, which ensures more rapid osmoadaptation of the cells (Wood, 1999). Although the accumulation of exogenous osmolytes is faster (the transporters are activated within a minute), one might wish to test the contribution of endogenous osmolytes to the stability and solubility of proteins in vivo. Exposing the cells to osmotic upshift without providing any exogenous osmolyte will force them to counteract the osmotic stress by the synthesis of endogenous osmoprotectants. We followed this procedure: E. coli BL21(DE3) cells transformed with pET16b plasmid bearing the tetra-Cys CRABP or P39A tetra-Cys CRABP under the T7 promoter are cultured at 37° in modified M9 minimal medium and preloaded with FlAsH as described earlier. At the time of induction (0.4 mM IPTG), the osmolality of the medium is increased by the addition of 300 mM sterile NaCl. The influence of the endogenously synthesized osmolytes (here trehalose) on the aggregation propensity is monitored by following FlAsH fluorescence of the bulk suspension at 530 nm (excitation 500 nm) at 37°.
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