Using a Xenopus Oocyte Protein Refolding Assay

John J. Heikkila, Angelo Kaldis, and Rashid Abdulle

Summary

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, microinjection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

Key Words: Aggregation; luciferase; microinjection; molecular chaperone; recombinant protein; refolding; small heat shock protein; Xenopus oocyte.

1. Introduction

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of cellular proteins under normal conditions and are upregulated when cells are exposed to environmental stress (e.g., elevated temperature, sodium arsenite, and heavy metals) (1-4). Hsps are composed of three major families, the high molecular weight (Hsp90), the Hsp70, and the small Hsp (SHsp) family. During environmental stress, SHsps bind to denatured or partially unfolded target proteins, prevent their aggregation, and maintain them in a soluble state until they can be refolded back into an active form by other molecular chaperones, including the Hsp70 family (5-11).

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

Protein aggregation is detrimental to a living cell and has been implicated in a variety of human diseases, including Alzheimer's disease, muscle myopathy, and multiple sclerosis (12,13). Various assays have been developed to monitor the ability of purified or recombinant SHsps to inhibit heat- or chemical-induced target protein aggregation in vitro using light scattering (14-18). The determination of whether an SHsp maintains its denatured target protein in a folding competent state has employed in vitro assays using either rabbit reticulocyte or wheat germ lysates as a source of molecular chaperones involved in refolding (15,19).

We developed an assay to assess the ability of a molecular chaperone, namely the Xenopus SHsp, Hsp30C, to maintain denatured client protein, firefly luciferase (LUC), in a folding competent state using microinjected Xenopus oocytes (19,20). Using the methods described here, we found that LUC when heat denatured and microinjected into oocytes regained very little enzyme activity. However, heat denaturation of LUC in the presence of Xenopus Hsp30C resulted in 100% enzyme reactivation following oocyte injection.

Xenopus oocytes contain a variety of molecular chaperones involved in protein folding, including Hsp70, but do not contain detectable levels of Hsp30 protein or messenger ribonucleic acid (RNA) (19,21-24). This in vivo refolding assay requires less molecular chaperone and substrate than in vitro refolding assays (19). Also, this protocol has been used successfully in our laboratory with SHsps from other organisms, including the American bullfrog Rana catesbeiana and the fruit fly Drosophila melanogaster. It is likely that this assay could be used with a wide variety of molecular chaperones. Coupling this assay with deletion or site-specific mutagenesis of the Hsp will allow the determination of protein domains essential for maintenance of the client protein in a folding-competent state (19,20).

2. Materials

1. pRSET vector and ProBond column system (Invitrogen; Burlington, ON, Canada).

2. Deoxyribonucleic acid (DNA) encoding Xenopus Hsp30C.

3. Isopropylthio-P-d-galactoside (IPTG).

4. Guanidinium lysis buffer: 6 M guanidinium hydrochloride, 20 mM sodium phosphate, and 500 mM sodium chloride, pH 7.8.

5. Protein dialysis buffer: 50 mM Tris-HCl at pH 8.0, 0.1 mM ethylenediaminetetraacetic acid (EDTA), and 25 mM NaCl.

6. Branson Sonifier 250 sonicator (VWR International; Mississauga, ON, Canada).

7. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) equipment.

8. Nanoject II oocyte microinjector with glass 3.5-in. (7.7-cm) capillary tubes (Drummond; Broomall, PA).

9. Penicillin and streptomycin.

10. Microsep 3K microconcentrator column (Pall Filtron; Northborough, MA).

11. Bicinchoninic acid (BCA) protein assay (Pierce; Rockford, IL).

12. Micropipet puller (Harvard Apparatus, Canada; Saint-Laurent, QC, Canada).

13. Variable-volume micropipeters (10 and 100 ||L).

14. Teflon homogenizer for 1.5-mL microfuge tubes.

15. Injection buffer: 50 mM HEPES-KOH at pH 7.5.

16. Mineral oil.

17. Modified Barth's solution (MBS): 88 mMNaCl, 1 mM KCl, 0.7 mMCaCl2, 1 mMMgSO4, 5 mM HEPES at pH 7.8, and 2.5 mM NaHCO3.

18. Ficoll.

19. Firefly LUC and luciferase assay reagent (Promega, Madison, WI).

20. Homogenization buffer: 5 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, 2 mM adenosine triphosphate (ATP), 2% protease inhibitor cocktail (Sigma, St. Louis, MO), and 125 mM HEPES-KOH at pH 7.5.

21. TD 20/20 luminometer (Turner Designs, Sunnyvale, CA).

22. Xenopus laevis females (Xenopus I, Ann Arbor, MI).

23. Dissecting microscope with fiber-optic lights.

3. Methods

The methods described outline the induction and purification of Xenopus Hsp30C recombinant protein as well as the protocol for the LUC enzyme reactivation assay using microinjected Xenopus oocytes.

3.1. Induction of Xenopus Recombinant Hsp30C Synthesis in Escherichia coli

Previously in our laboratory, the reading frame of Xenopus Hsp30C was cloned into a pRSETB (Invitrogen) expression vector that contains an N-terminal 6x-histi-dine tag (18) (see Note 1). The resultant Hsp30C-pRSETB vector was transformed into Escherichia coli BL21 (DE3) cells and stored as a glycerol stock at -80°C. The following protocol details the induction of recombinant Hsp30C expression with IPTG and purification of the resultant protein using a ProBond nickel affinity column (18,25-27):

1. Grow the transformed E. coli cells at 37°C in ZB media ((1% tryptone, 0.5% NaCl) overnight.

2. Inoculate 50 mL M9ZB media with 2.5 mL of the overnight culture and grow to an optical density of 0.6 to 0.8 at 600 nm.

3. Add IPTG to a final concentration of 0.4 mM and incubate for 4 h to induce Hsp30C gene expression (see Note 2).

4. Centrifuge the cells at 12,000g for 30 min at 4°C. Discard the supernatant, freeze the pellet in liquid nitrogen, and then thaw quickly at 37°C. Repeat the freezing and thawing step twice.

5. Resuspend the cells in 5 mL guanidinium lysis buffer (see Note 3).

6. Agitate the solution on an orbital shaker for 4 h at 22°C.

7. Place the tubes on ice and sonicate with 20 sets of three 1-s bursts (Output = 5; Duty cycle = 80%).

8. Centrifuge the sonicated mixture at 3000g at 4°C for 30 min, transfer the supernatant or bacterial lysate to a new tube, and store at -80°C.

3.2. Purification of Recombinant Hsp30C

1. The ProBond beads and columns are prepared and equilibrated with 5 mL denaturing buffer at pH 7.8 (see Note 3) according to manufacturer's instructions. Prior to use, the column is centrifuged, and the supernatant is discarded.

2. Add 5 mL bacterial lysate to the column and mix gently by inversion (see Note 4).

3. Centrifuge at 600g for 5 min and remove the unbound lysate.

4. Add 5 mL denaturing buffer at pH 7.8 to the column and mix gently by inversion.

5. Place the column on an orbital nutator and agitate for 2 min at room temperature.

6. Centrifuge at 1200g for 3 min and discard the supernatant.

pRSETB 30C 30C

Oh 4h Oh 4h purified

Fig. 1. Expression and purification of Hsp30C recombinant protein from E. coli. Total bacterial protein from E. coli BL21 (DE3) cells containing either the expression vector (pRSETB) or the vector with the Hsp30C gene insert (30C) were collected either before or after 4 h of IPTG addition. Protein was analyzed by Coomassie brilliant blue staining. Purified 30C after nickel affinity column chromatography is shown. The asterisks indicate the location of 30C. Molecular mass markers in kilodaltons are indicated on the left side of the figure.

7. Repeat steps 5 to 7 three times each with 5 mL denaturing buffer at pH 6.0 and denaturing buffer at pH 5.3 and then twice with denaturing buffer at pH 5.0 (see Note 5).

8. After the last centrifugation, clamp the ProBond column in a vertical position and snap off the tip to allow the excess buffer to drain.

9. Cap the tip of the column and add 5 mL denaturing buffer at pH 4.0 and mix gently by inversion.

10. Centrifuge at 1200g for 30 s to pack the resin.

11. Clamp the column in a vertical position, remove the cap from the tip, and collect 1-mL fractions.

12. Repeat steps 10 to 12 and freeze all fractions at -80°C.

13. Analyze 20 ||L of each fraction by means of SDS-PAGE and staining with Coomassie brilliant blue.

14. Combine all fractions that contain the recombinant protein and dialyze for 15 h against protein dialysis buffer.

15. Concentrate the dialysate (~5-10 mL) using a 3K Microsep microconcentrator column (see Note 6) to the desired volume (200-300 |L) and transfer to a microcentrifuge tube. Take a sample for SDS-PAGE to check the purity of the protein preparation (Fig. 1) and then store in aliquots at -80°C.

16. Determine the concentration of the protein using a BCA protein assay.

Fig. 1. Expression and purification of Hsp30C recombinant protein from E. coli. Total bacterial protein from E. coli BL21 (DE3) cells containing either the expression vector (pRSETB) or the vector with the Hsp30C gene insert (30C) were collected either before or after 4 h of IPTG addition. Protein was analyzed by Coomassie brilliant blue staining. Purified 30C after nickel affinity column chromatography is shown. The asterisks indicate the location of 30C. Molecular mass markers in kilodaltons are indicated on the left side of the figure.

3.3. Xenopus Oocyte Isolation and Microinjection Apparatus

Xenopus oocytes are isolated according to standard procedures and maintained in covered Petri dishes containing MBS media with 100 U/mL penicillin and 100 |g/mL streptomycin at 22°C. Only normal-appearing oocytes that have been incubated overnight should be used in this assay. Microinjection of protein is carried out with a Nanoject II system set up to deliver a volume of 27.6 nL according to manufacturer's instructions (see Note 7).

Microinjection needles or micropipets are made from glass capillary tubes using a micropipet puller. The tips of the needles are broken off with a pair of fine forceps and stored until needed. New microinjection needles are used for each sample. The placement of 10 to 20 |L of a sample on a strip of parafilm facilitates the filling of the micropipet with the Drummond Nanoject II. Prior to microinjection, oocytes were transferred to fresh MBS media containing 4% Ficoll to minimize cytoplasmic leakage after injection.

3.4. Luminometer Settings

The TD 20/20 luminometer is used to measure the production of light resulting from the enzymatic action of LUC on luciferin present in the luciferase assay reagent. This equipment is used according to the manufacturer's instructions employing a 12 mm adapter to hold 1.5 ml clear microcentrifuge tubes with the Sensitivity Adjust set to 100.

3.5. Preparation of LUC or Hsp30C Samples for Microinjection

Dilute a sample of the stock firefly LUC solution to 2.5 ||M with injection buffer and store 4-|L aliquots at -80°C. Prepare recombinant Hsp30C by diluting it to concentrations ranging from 16.7 to 100.2 |M (depending on the final molar ratio of Hsp30C to LUC desired, namely, 5/1 to 30/1) using protein dialysis buffer and store at -80°C until needed.

3.6. Activity of Nondenatured LUC After Oocyte Injection

1. Place a microcentrifuge tube on ice with 24 ||L of injection buffer (see Note 8).

2. Add 1.0 ||L LUC to the tube followed by mixing and brief centrifugation at 12,000g for 20 s.

3. Incubate the solution at 22°C for 10 min in the dark (covered in aluminum foil).

4. Add 75 |L injection buffer and mix as above.

5. Transfer 20 ||L of the sample onto parafilm and use the Drummond Nanoject II settings to fill the microinjection needle.

6. Place 10 oocytes into a dish for microinjection containing MBS with 4% Ficoll.

7. Inject 27.6 nL of the LUC mixture per oocyte.

8. Immediately after injection, divide the oocytes between two microcentrifuge tubes (five each) and remove all of the oocyte media using a Pasteur pipet.

9. Add 20 |L freshly prepared homogenization buffer to each tube and homogenize with a Teflon pestle made for microcentrifuge tubes.

10. Vortex each tube for 5 s and centrifuge at 12,000g for 5 min.

11. Take 2 |L of the clear supernatant, taking care to avoid the pellet and overlying pellicle, and transfer it to a 1.5-mL clear microcentrifuge tube.

12. Add 18 |L LUC reagent equilibrated at room temperature to the 2 |L oocyte extract and mix.

Fig. 2. LUC heat denatured in the presence of Hsp30C can be refolded in vivo after microinjection into Xenopus oocytes. LUC (0.2 |lM) was incubated at 22°C (♦) or heat denatured alone (X) or in the presence of either 6 |M BSA (•) or 30C at 30C:LUC molar ratios of 1/1 (□), 10/1 (A), or 30/1 (A). Mixtures (containing 1.38 fmol of LUC in 26.7 nL) were microinjected into Xenopus oocytes, and LUC activity in the oocytes was monitored over time. Data are representative of three to five trials and are shown as the mean ± standard error.

Fig. 2. LUC heat denatured in the presence of Hsp30C can be refolded in vivo after microinjection into Xenopus oocytes. LUC (0.2 |lM) was incubated at 22°C (♦) or heat denatured alone (X) or in the presence of either 6 |M BSA (•) or 30C at 30C:LUC molar ratios of 1/1 (□), 10/1 (A), or 30/1 (A). Mixtures (containing 1.38 fmol of LUC in 26.7 nL) were microinjected into Xenopus oocytes, and LUC activity in the oocytes was monitored over time. Data are representative of three to five trials and are shown as the mean ± standard error.

13. Immediately place the tubes into the TD 20/20 luminometer and take readings for a period of 12 s following a 3-s delay. The average of the duplicate sample readings should be approx 800 to 1000 cpm or more and will be set as the 100% value against which all remaining samples will be compared.

3.7. Activity of Heat-Denatured LUC After Oocyte Injection

The next sample will determine the loss of LUC enzyme activity after heat denatur-ation and microinjection into oocytes.

1. Place a microcentrifuge tube on ice with 24 |L injection buffer.

2. Add 1.0 |L LUC to the tube, followed by mixing and brief centrifugation at 12,000g for 20 s.

3. Cover the tubes with aluminum foil and incubate at 42°C for 8 min, followed by 2 min at 22°C (see Note 9).

4. Follow Subheading 3.6., steps 4 to 13. The luminometer readings of this time-point are expressed as a percentage of nondenatured LUC value obtained in Subheading 3.6. Ideally, this time point should be 1 to 8% of the nondenatured LUC sample assayed previously. Furthermore, incubation of injected oocytes at 22°C for time periods ranging up to 100 min should show minimal LUC activity (Fig. 2).

Xenopus Oocyte

Fig. 3. LUC heat denatured in the presence of 30C and N-30C but not C-30C can be refolded in Xenopus oocytes. LUC (0.2 ||M) was heat denatured alone (X) or with 6.0 ||M of 30C (O), N-30C (□), or C-30C (▲) at 42°C. Samples containing 1.38 fmol of LUC in 26.7 nL were microinjected into Xenopus oocytes, and LUC activity was determined over time as indicated in Subheadings 2. and 3. Data are representative of three to five trials and are shown as the mean ± standard error.

Time (min)

Fig. 3. LUC heat denatured in the presence of 30C and N-30C but not C-30C can be refolded in Xenopus oocytes. LUC (0.2 ||M) was heat denatured alone (X) or with 6.0 ||M of 30C (O), N-30C (□), or C-30C (▲) at 42°C. Samples containing 1.38 fmol of LUC in 26.7 nL were microinjected into Xenopus oocytes, and LUC activity was determined over time as indicated in Subheadings 2. and 3. Data are representative of three to five trials and are shown as the mean ± standard error.

3.8. Enzyme Activity of LUC Heat Denatured in the Presence of Hsp30C

This sample will determine whether Hsp30C can maintain heat-denatured LUC in a folding-competent state such that it can regain enzyme activity after injection of the complexes into Xenopus oocytes.

1. Place a microcentrifuge tube on ice with 22.5 |L injection buffer.

3. Add 1.0 |L LUC to the tube, followed by mixing and brief centrifugation at 12,000g for 20 s.

4. Cover the tubes with aluminum foil and incubate at 42°C for 8 min followed by 2 min at 22°C.

5. Follow Subheading 3.6., steps 4 to 13. However, with this sample it is essential to follow the enzyme activity of LUC over a period of 100 min at 22°C after injection into oocytes. The suggested incubation times are 0, 20, 40, 60, 80, and 100 min using a total of 10 oocytes per time-point. The injections of the oocytes are staggered to facilitate LUC enzyme analysis. The luminometer readings of these time-points are expressed as a percentage of nondenatured LUC value obtained in Subheading 3.6. A typical LUC enzyme reactivation curve is shown in Fig. 2. As mentioned in Subheading 1.1., this assay can also be used to examine the various protein domains associated with Hsp30C to maintain LUC in a folding-competent state (19). As shown in Fig. 3, deletion of 17 amino acids from the N-terminal end (N-30C) has no effect on LUC enzyme reactivation in Xenopus oocytes, but removal of 25 amino acids from the C-terminal end (C-30C) dramatically reduces the molecular chaperone function of Hsp30C.

4. Notes

1. As described in a previous publication, the entire open reading frame of Xenopus Hsp30C was obtained by polymerase chain reaction such that a BamHl restriction site was created just 5' to the start codon and an EcoRI restriction site just 3' to the translation stop codon (18). This fragment was then cloned into a pRSETB vector. This type of approach can be used with any molecular chaperone gene, but it is essential that the chimeric plasmid is sequenced to ensure no mutations and that the coding sequence is in the correct reading frame.

2. With other molecular chaperones, it is necessary to determine the optimal conditions for recombinant protein induction. Preliminary experiments should vary the IPTG concentration from 0.4 to 1.0 mM for periods of time ranging from 2 to 10 h, followed by analysis by SDS-PAGE and Coomassie brilliant blue staining.

3. Because the pH of the guanidinium lysis buffer and denaturing buffer solutions tend to drift with time, they should be checked prior to use. In our experience, the inability to recover recombinant protein from the ProBond nickel affinity column can often be traced to incorrect pH of the denaturing buffer.

4. It is important not to exceed the binding capacity of the nickel affinity resin, which is between 1 and 5 mg/mL.

5. The application of denaturing buffer (pH 5.0) to the column may result in the elution of some bound SHsp from the resin, but this step is necessary to obtain a pure sample of Hsp30C. The ProBond beads can be reused at least two more times after the column has been recharged following manufacturer's instructions.

6. The Microsep Concentrator column can be reused to concentrate additional batches of Hsp30C protein by adding 2 mL of 20% ethanol onto the filter and storing it at 4°C until required.

7. Microinjection of 1.38 fmol of LUC plus 5- to 30-fold Hsp30C in 26.7 nL into oocytes appears to be optimal. Injection of higher amounts of LUC (e.g., 10.12 fmol) and accompanying SHsp results in reduced enzyme reactivation (19). It is likely that higher amounts may overload the available chaperone folding machinery in oocytes.

8. Because this assay is very sensitive, it is essential that all pipeters are calibrated on a regular basis.

9. The time required for heat denaturation of LUC at 42°C may vary between lot numbers of enzyme. Although our normal denaturation time is 8 min, we have found that with some batches of LUC, a longer heat denaturation time up to 15 min may be required to achieve a final enzyme activity of 1 to 8%.

Acknowledgments

This research was supported by Natural Science and Engineering Research Council grants to J. J. H., who is also the recipient of a Canada Research Chair in Stress Protein Gene Research.

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