Purifying Natively Folded Proteins...rkosyl, Triton X ...

3 downloads 192 Views 1MB Size Report
CHAPS also worked well (data not shown). All three ... recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ..... USB connectivity and.
Benchmarks

Benchmarks Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS Hu Tao1, Wenjun Liu1, Brandi N. Simmons1, Helen K. Harris1, Timothy C. Cox2, and Michael A. Massiah1 1Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA and 2Division of Craniofacial Medicine, Department of Pediatrics, University of Washington, Seattle, WA, USA BioTechniques 48:61-64 (January 2010) doi 10.2144/000113304 Keywords: protein solubilization; inclusion bodies; protein purification; sarkosyl; detergents

We describe a rapid, simple, and efficient method for recovering glutathione S-transferase (GST)- and His6-tagged maltose binding protein (MBP) fusion proteins from inclusion bodies. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized >95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS. We demonstrate for the first time that this combination of three detergents significantly improves binding efficiency of GST and GST fusion proteins to gluthathione (GSH) Sepharose. Proteins are usually engineered to be overexpressed in Escherichia coli as fusion proteins, commonly with glutathione S-transferase (GST) (1), His6 tag (2,3), small ubiquitinlike modifier (SUMO) (4,5), thioredoxin (6), and maltose binding protein (MBP) (3,7). These fusion systems promise increased solubility of target proteins, except for the His6 tag, with single-step purification efficiency. However, many recombinant proteins, especially those of eukaryotic origin, aggregate or become packaged into inclusion bodies (8–11). Refolding recombinant proteins from inclusion bodies can be challenging and yields of correctly folded proteins can be low (2,12). We observed a number of proteins that readily formed inclusion bodies in E. coli, even with optimized conditions. The problem is amplified when the cells are grown in M9 minimal media, which is required to isotopically label proteins for nuclear magnetic resonance (NMR) and x-ray crystallographic studies. The increased insolubility when using M9 minimal media compared with Luria Bertani (LB) media may be the result of differences in the cellular environments of overexpressed proteins, or may be proteinspecific (13,14). Vol. 48 | No. 1 | 2010

Lysis buffers containing 0.3–2% sarkosyl (Cat. no. 61207-5000; Acros, Morris Plains, NJ, USA) have been used to solubilize GST and other proteins expressed in bacteria grown in LB media (10,11,15–17), but are less effective when using minimal media. Three cysteine-rich zinc binding domains (RING, Bbox1, and Bbox2) from Midline1 (MID1), a microtubule-associated ubiquitin E3 ligase, readily form inclusion bodies when expressed as GST fusion proteins in M9. We found these typical amounts of sarkosyl were insufficient to solubilize these GST fusion proteins and a related MBP fusion protein, MBP-RBCC (RING-Bboxcoiled-coil domains of MID1) (Figure 1A). Therefore, we tried higher percentages of sarkosyl (up to 10%) in the lysis buffer, and obtained 40–70% of the GST fusion proteins in the solubilized extract based on the intensities of the protein band following SDS-PAGE. However, we determined that soaking the insoluble pellet (containing essentially 100% insoluble GST fusion RING, Bbox1, Bbox2, or MBP-RBCC) from 5–10 g lysed cells in 2 mL ST buffer (50 mM Tris, 300 mM NaCl, 5 mM ZnCl2 , 10 mM β-mercaptoethanol) with 10% (w/v) sarkosyl for 6–24 h effectively and efficiently solubilized >95% of the

61

proteins from the pellet (Figure 1, A and B). We found 10% sarkosyl to be optimal because higher concentrations were too viscous, leading to difficulty in subsequent purification steps. The solubilizing effects of the sarkosyl actually decreased when the concentration of sarkosyl was >10% (data not shown). This simple approach of using 10% sarkosyl was effective in solubilizing at least six different proteins tested, all of which formed inclusion bodies even when fused to His6 -MBP and His6 tag. Of note, we observed that the majority of other proteins found in the pellet were also solubilized with 10% sarkosyl (Figure 1, A and B), suggesting that this methodology is broadly applicable. Even though sarkosyl can solubilize GST fusion proteins, purifying these proteins in the presence of the detergent can be challenging and difficult (12). Consistent with previous reports (10,11,15), we observed that GST and our GST fusion proteins could not be affinity-purified even in 0.3% sarkosyl. To overcome the problem of high sarkosyl concentrations, the 10% sarkosyl–solubilized pellet solution was diluted with the lysate to yield a 2% sarkosyl solution, or to 1% with a variety of common buffers. In each case, solubility of the overexpressed protein was maintained, although at lower sarkosyl concentrations (95% soluble GST-Bbox1 (lane 8). (B) Similar results as GST-Bbox1 were observed with His6-MBP-RBCC (RING-Bbox1-coiled-coil domains from MID1). (C) Binding of GST-Bbox1 to GSH Sepharose. Equal amounts of GST-Bbox1 in the presence of the indicated detergents was used for the binding. The same amount of the resin of each sample was used for SDS-PAGE. GST-Bbox1 bound the GSH Sepharose best when all three detergents were present at the determined ratio. (D) Solubilization and purification of the His6-tagged ThuB oxidoreductase. Approximately 30% of the protein was found in the crude lysate without sarkosyl in the lysis buffer. Lane 3 shows the supernatant of the 5% sarkosyl-soaked pellet; insoluble ThuB in the pellet support the observation that 10% sarkosyl was optimal for solubilizing >95% of the protein. Lane 5 shows His6-ThuB bound the Ni2+ resin in the presence of 1% sarkosyl.

A

B

C

Figure 2. Native folding of detergent solubilized proteins. (A) The enzymatic activity of purified GST (from horse liver) was measured in the presence of sarkosyl with increasing amounts of Triton X-100 and CHAPS to emphasize how GST behaves in the three detergents. Colorimetric changes, as result of formation of a GSH-CNDB adduct, was measured at 340 nm. (B) Superposition of a portion of the 1H-15N HSQC spectra of 15N-labeled MID1 Bbox2 in the presence (red) and absence (green) of 1% sarkosyl. The full spectrum in the absence of detergent is shown in panel C (green). The weaker signals of the spectrum in red indicate slower Bbox2 tumbling rates, mostly likely due to sarkosyl molecules encapsulating the protein. (C) Superposition of the 1H-15N HSQC spectra of Bbox2 in the presence (red) and absence (green) of 1% sarkosyl, 2% Triton X-100 and 20 mM CHAPS. Bbox2 remained natively folded in the presence of all three detergents. Adding either Triton X-100, or CHAPS had no effect on the NMR spectra of free Bbox2 or Bbox2 with 1% sarkosyl.

turer’s specification. The formation of GSH-CDNB adduct was measured by absorbance at 340 nm for 30 min. Detergents, either alone or in combinations of different ratios, were added to fresh assay solution to determine their effect on GST activity. In the presence of 0.3% sarkosyl— the least amount of sarkosyl used in previous reports (10,11,15–17)—GST was inactive. Addition of 1% Triton X-100 and 10 mM CHAPS to GST with 1% sarkosyl rescued ~10% of the original activity, while 2% Triton X-100 and 20 mM CHAPS regained ~60% of GST activity and 3% Triton X-100 and 30 mM CHAPS yielded ~80% of the original enzymatic activity. Individually, CHAPS and Triton X-100 at these concentrations could only recover ~30–40% of the GST activity. In the control experiment, 1% Triton X-100 and 10 mM CHAPS, without sarkosyl, did not affect GST enzymatic activity. GSH Sepharose binding was also significantly enhanced in the presence of all three detergents with a binding affinity estimated to Vol. 48 | No. 1 | 2010

be three- to five-fold better in the three detergents compared with free GST. We postulate that the sarkosyl molecules encapsulate proteins and disrupt aggregates. Triton X-100 and CHAPS, with critical micelle concentrations of 0.25 mM and 6–10 mM, respectively, form large mixed micelle or bicelle structures that incorporate sarkosyl molecules from the solution. In doing so, they decrease the apparent concentration of sarkosyl surrounding GST, potentially freeing active sites or facilitating proper protein refolding. The >80% yield of properly refolded proteins is significant when compared with yields obtained with other commonly employed methods. Two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC) NMR spectroscopy (Varian Inova 600 MHz spectrometer, VarianInc, Palo Alto, CA, USA) was used to gain further insight into each detergent’s mechanism of action. We observed that the 1H-15N signals of 15N-labeled MID1 Bbox2 were weaker

62

in the buffer with 1% sarkosyl compared with those without detergents (Figure 2B). The weaker signals are due to increased resonance line broadening from the slower isotopic molecular tumbling rate of Bbox2, likely the result of being encapsulated by sarkosyl molecules. Viscosity was ruled out as a potential cause because the addition of 2% Triton X-100 and 20 mM CHAPS, which would have increased viscosity, resulted in 1H-15N signal intensities returning to values similar to those of detergent-free Bbox-2 (Figure 2C). The lack of large chemical shift changes that would imply a drastic structural change or collapsed peaks that would indicate unfolding of Bbox2 suggests that the tertiary structure of Bbox2 (a ββα-RING fold with two coordinated zinc ions) was intact in the presence of 1% sarkosyl. The signals of the 2-D HSQC spectra of GST and MBP showed more collapsed signals in the presence of 1–2% sarkosyl, consistent with a molten globule state but not that of a denatured protein (data www.BioTechniques.com

Benchmarks

not shown). The spectra of these proteins in the presence of 1% sarkosyl, 2% Triton X-100, and 20 mM CHAPS were similar to natively folded GST and MBP. It is important to note that proteins denatured by urea, guanidine hydrochloride, or heat could not be refolded with just these three detergents. An intrinsically unstructured protein also remained unstructured in the three detergents (data not shown). As the ingredients for minimal media are relatively expensive and yet essential for isotopically labeling proteins for structural studies, it is important to maximize, in milligrams amounts, the yield of soluble folded protein. We therefore tested the protocol with one His6 -tagged and seven His6 -MBP fusion proteins, including one that contained two disulfide bonds. Incubation of the His6-tagged FMN/NAD-dependent trehalose oxidoreductase from Sinorhizobium meliloti (His6-ThuB) with either 5% or 10% sarkosyl resulted in >75% (Figure 1D) and >95% soluble protein (data not shown), respectively. Subsequent dilution to 1% sarkosyl enabled efficient affinity purification of His6-ThuB with Ni2+ resin (Cat. no. 30410; Qiagen, Valencia, CA, USA). Similarly, the interleukin binding protein with disulfide bonds was also successfully folded in the presence of all three detergents (data not shown). While some of the His6-MBP fusion proteins required the pellets to be incubated with 10% sarkosyl, others were soluble with 1% sarkosyl in the lysis buffer. Even though the His6-MBP fusion proteins could be purified with Ni2+ resin in the presence of 1% sarkosyl, the addition of Triton X-100 and CHAPS increased the binding. Based on our NMR spectra of solubilized protein purified in this manner, we believe it is important to have all three detergents to maximize yields.

Outstanding Speed Heating and cooling rates up to 5 ºC/s • No overshoots and quick stabilization at programmed temperature •

Highest Precision •

High block homogeneity and linear temperature gradients

Simplest Operation TFT display and mouse controlled operation • USB connectivity and LAN networking •

Highest Quality and Greatest Lifetime ISO 9001 and 13485 certi�ed manufacturing in Germany • Peltier elements with Long Life Technology •

Sample Flexibility 96-well Universal block compatible with 0.2 and 0.5 mL tubes or 96-well plates, 384-well block also available • High Pressure Lid option available for low volume PCR •

Acknowledgments

We thank Brian Krumm for helpful discussions, and for successfully applying our sarkosyl protocol to his His6 -MBP-ILC4S protein. This work was supported in part by the Oklahoma State University Agricultural Experimental Station (Project no. 2527) and the NSF CAREER (no. 0546506) grants.

Competing interests

The authors declare no competing interests.

References 1. Smith, D.B. and K.S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40. 2. Ausubel, F., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1995. Short Protocols in Molecular Biology. John Wiley & Sons. 3. Pryor, K.D. and B. Leiting. 1997. High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein doubleaffinity fusion system. Protein Expr. Purif. 10:309-319. 4. Butt, T.R., S.C. Edavettal, J.P. Hall, and M.R. Mattern. 2005. SUMO fusion technology for difficult-to-express proteins. Protein Expr. Purif. 43:1-9. 5. Marblestone, J.G., S.C. Edavettal, Y. Lim, P. Lim, X. Zuo, and T.R. Butt. 2006. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 15:182-189. 6. LaVallie, E.R., E.A. DiBlasio, S. Kovacic, K.L. Grant, P.F. Schendel, and J.M. McCoy. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio./ technology (Nature Publishing Company) 11:187-193. 7. Kapust, R.B. and D.S. Waugh. 1999. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8:1668-1674. 8. Przybycien, T.M., J.P. Dunn, P. Valax, and G. Georgiou. 1994. Secondary structure characterization of beta-lactamase inclusion bodies. Protein Eng. 7:131-136. 9. Hahn, A.W., S. Regenass, F. Kern, F.R. Buhler, and T.J. Resink. 1993. Expression of soluble and insoluble fibronectin in rat aorta: effects of angiotensin II and endothelin-1. Biochem. Biophys. Res. Commun. 192:189-197. 10. Frangioni, J.V. and B.G. Neel. 1993. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal. Biochem. 210:179-187. 11. Frankel, S., R. Sohn, and L. Leinwand. 1991. The use of sarkosyl in generating soluble protein after bacterial expression. Proc. Natl. Acad. Sci. USA 88:1192-1196. 12. Lilie, H., E. Schwarz, and R. Rudolph. 1998. Advances in refolding of proteins produced in E. coli. Curr. Opin. Biotechnol. 9:497-501. 13. Arakawa, T. and S.N. Timasheff. 1985. The stabilization of proteins by osmolytes. Biophys. J. 47:411-414. 14. Bogdanov, M. and W. Dowhan. 1999. Lipid-assisted protein folding. J. Biol. Chem. 274:36827-36830. 15. Burgess, R.R. 1996. Purification of overproduced Escherichia coli RNA polymerase sigma factors by solubilizing inclusion bodies and refolding from Sarkosyl. Methods Enzymol. 273:145-149. 16. Marskak, D.R. 1996. Strategies for Protein Purification and Characterization: a Laboratory Course Manual. CSH Laboratory Press, Cold Spring Harbor, NY. 17. Zhuo, Q., J.H. Piao, R. Wang, and X.G. Yang. 2005. Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies. Protein Expr. Purif. 41:53-60. Received 12 March 2009; accepted 15 October 2009. Address correspondence to Michael A. Massiah, Department of Biochemistry and Molecular Biology, Oklahoma State University, 246 Noble Research Center, Stillwater, OK 74078, USA. email: [email protected]

PEQLAB US • Wilmington, DE 19810 Toll-Free (US): 877 737 5220 [email protected] • www.peqlab.us

64

www.BioTechniques.com