Preparation of functional recombinant protein from E. coli using a nondetergent sulfobetaine.

Expression of foreign proteins in E. coli is frequently stymied by the formation of insoluble inclusion bodies. To recover the insoluble protein, the inclusion body is usually solubilized with a denaturing reagent such as urea and guanidine HCl, followed by dialysis to renature the target protein. Nondetergent sulfobetaines (NDSB) are a family of zwitterionic compounds carrying a sulfobetaine hydrophilic group and a hydrophobic group that is not long enough for micelle formation. Recently, NDSBs have been used as the solubilizing and stabilizing reagents for protein purification and renaturation (2,6–8). Our previous trials on preparation of GST-C/EBPβ from E. coli did not recover active GST-C/EBPβ using either the Sarkosyl detergent (4) or the regular denaturation-renaturation protocol (1). In this study, we tested the effect of NDSB 201 on renaturation of GST-C/EBPβ purified from E. coli and found that NDSB 201 facilitates the preparation of active recombinant GSTC/EBPβ from inclusion bodies. To express GST-C/EBPβ fusion protein, cDNA encoding the rodent C/ EBPβ was placed downstream of the GST fragment in pGEX5T-1. The resultant construct was transformed into BL21(DE3)pLysS for protein expression. A positive colony was inoculated into 25 mL LB medium containing 100 μg/mL ampicillin and incubated at 37°C overnight. Next day, the saturated culture was placed into 500 mL culture medium and incubated at 37°C until the cell density reached A600 = 0.8. Protein expression was induced with 0.5 mM IPTG at 37°C for 2 h. The bacteria were spun down and resuspended in the lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 1% Triton X-100, and 5 mM DTT). Cells were lysed by one cycle of freezing and thawing plus a brief sonication in the presence of the complete, EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). The lysate was then treated with 20 μg/mL DNase I at 37°C for 1 h. The insoluble inclusion body was spun down and washed twice with 1% Triton X-100. The inclusion body was dissolved in the denaturation solution (6 M guanidine HCl, 25 mM DTT, and 50 mM Tris, pH 7.5) at 4°C for 1 h. The protein concentration of solubilized sample was then adjusted to 1 μg/μL with the denaturation solution. For renaturation, the solubilized sample was quickly diluted with a tenfold volume of renaturation solution (1 M NDSB, 0.2 M NaCl, 1 mM DTT, and 50 mM Tris, pH 7.5) and incubated at 4°C for 1 h. The renatured sample was dialyzed against double-distilled water at 4°C overnight. The dialyzed sample was concentrated with PEG 20 000 and purified with glutathione agarose. The purified GST-C/EBPβ fusion protein was eluted with the elution buffer (20 mM reduced glutathione, 120 mM NaCl, and 100 mM Tris, pH 8.0), dialyzed against 20 mM Tris (pH 7.5), and stored at -20°C. We tested the efficiency of NDSB 201 in the renaturation of GST-C/ EBPβ. We reasoned that correctly folded GST-C/EBPβ fusion protein will bind to the glutathione agarose beads and be eluted with the reduced glutathione. As shown in Figure 1A, the eluted GST-C/EBPβ analyzed by SDSPAGE appeared to match the predicted size. To further test the bioactivity of the renatured GST-C/EBPβ, we performed the electrophoretic mobility shift assay (EMSA) and the GST pulldown assay. For EMSA, a C/EBPβ responsive element (FPII, 5′-TTGTTGCTCAACATGTTGA-3′) derived from the rat pregnancy-specific glycoprotein gene, rnCGM3, was used as the probe (3). The radiolabeled FPII fragment was incubated with C/EBPβ translated in vitro and the renatured GST-C/ EBPβ, respectively. As shown in Figure 1B, lanes 3 and 7, a specific DNA-protein complex was formed with both protein samples because unlabeled FPII could compete with complex formation (lanes 2 and 6). Moreover, antibody specific to C/EBPβ was able to abolish the formation of the complex between FPII and the C/EBPβ translated in vitro or the renatured GST-C/EBPβ (Figure 1B, lanes 4 and 8). Antibody specific to GST was also able to prevent the formation of the FPII-GST-C/EBPβ complex (Figure 1B, lane 5). Because C/EBPβ interacts with the p50 subunit of NFκB (5), we also performed pulldown assays using the renatured GSTC/EBPβ and 35S-methionine-incorporated p50. As shown in Figure 1C, p50 specifically interacted with GST-C/ EBPβ (lane 3) but not GST (lane 2). These results suggested that preparation of active GST-C/EBPβ from inclusion Benchmarks