Editing HRV 3C Expression and Purification

== Protocol 1, Autoinduce<br>  ==

1. transform pET28b-Cold-Native 3C construct into E. coli BL21 Star (DE3) pRARE2. Plate out on LB-agar containing kanamycin (30 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C.<br> 

<br>The BL21 Star (DE3) pRARE2&nbsp; strain is derived from BL21 Star (DE3), transformed with &nbsp;pRARE2 plasmid from Rosetta2 (DE3).<br> 

2.&nbsp; Pick 2 colonies from the plate to inoculate 2 x 4 ml LB medium containing kanamycin (30 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C in a shaker/incubator.<br> 

3. Use the overnight cultures to inoculate 2 x 1 L ZYM 5052 autoinduction medium containing kanamycin (100 μg/ml) and chloramphenicol (33 μg/ml) in 2-L flasks.<br> 

<br>4. Grow the cultures at 37°C until the OD600 is approx. 1. Then lower the temperature of the shaker/incubator to 20°C and incubate the cultures overnight.<br><br> 

5. Harvest the cells by spinning for 5 min at 6,000 rpm using a S3 rotor.<br> 

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6. Resuspend the pellet in 40 ml lysis buffer. <br> 

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7. Sonicate (2 x 3 min on ice-water) to lyse the cells.<br> 

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8. Spin the cell lysate for 15 min at 12,000 rpm. Spin again at 25000 rpm at 20 min.<br> 

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9. Load the supernatant to a 5-ml Ni column.<br> 

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10. Wash the column with washing buffer until no more protein elutes (monitored by the absorbance at 280 nm).<br> 

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11. Elute the protein with elution buffer (150 mM imidazole).<br> 

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=== For GST-3C  ===

<br>9. Load the supernatant to a 5-ml GSTrap column equilibrated with wash buffer.<br>10. Wash the column with wash buffer until no more protein elutes (monitored by the absorbance at 280 nm).<br>11. Elute the protein with elution buffer. Collect the eluate in 2-ml fractions.<br> 

<br>12. Pool the protein containing fractions and dialyze overnight against 50 ml storage buffer at 4°C.<br> 

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13. For product: dilute the final solution with storage buffer to a final concentration of 1 mg/ml and store in 0.5-ml aliquots at -80°C.<br> 

<br>The amount of His6-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 0.253 (a His6-3C protease solution of 1 mg/ml gives an A280 of 0.253). The molecular weight of His6-3C protease is 21.7 kDa.<br> 

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The amount of GST-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 1.005 (a His6-3C protease solution of 1 mg/ml gives an A280 of 1.005). The molecular weight of His6-3C protease is 46.4 kDa.<br> 

=== <br>  ===

=== ZYM-5052 medium for auto-induction<br>  ===

958 ml ZY<br>20 ml 50 x M<br>20 ml 50 x 5052<br>2 ml 1 M MgSO4<br>0.2 ml 1000 x trace elements<br> 

<br> 

=== ZY<br>  ===

tryptone&nbsp; 1%<br>yeast extract&nbsp; 0.5%<br> 

=== <br>  ===

=== 50 x M<br>  ===

Na2HPO4&nbsp; 25 mM<br>KH2PO4&nbsp; 25 mM<br>NH4Cl&nbsp; 50 mM<br>Na2SO4&nbsp; 5 mM 

=== <br>  ===

=== 50 x 5052<br>  ===

glycerol&nbsp; 0.5%<br>glucose&nbsp; 0.05%<br>alpha-lactose&nbsp; 0.2% 

<br> 

=== 1000 X Trace elements  ===

<br>50 mM FeCl3<br>20 mM CaCl2<br>10 mM MnCl2<br>10 mM ZnSO4<br>2 mM CoCl2<br>2 mM CuCl2<br>2 mM NiCl2<br>2 mM Na2MoO4<br>2 mM Na2SeO3<br>2 mM H3BO3<br><br><br> 

=== 500 x MgSO4  ===

<br>1 M MgSO4-7H2O<br>24.65 g/100 ml<br><br><br>Preparation of 100 ml 1000x trace elements stock solution (in ~60 mM HCl).<br>Prepare stock solutions of the salts (except FeCl3) as mentioned in the table below.<br>Dissolve 0.1 M FeCl3 in 50 ml of 100x diluted concentrated HCl.<br>Add to 36 ml sterile water:<br>50 ml&nbsp; 0.1 M FeCl3-6H2O&nbsp; 2.70 g/100 ml<br>2 ml&nbsp; 1.0 M CaCl2-2H2O&nbsp; 15.8 g/100 ml<br>1 ml&nbsp; 1.0 M MnCl2-4H2O&nbsp; 19.8 g/100 ml<br>1 ml&nbsp; 1.0 M ZnSO4-7H2O&nbsp; 28.8 g/100 ml<br>1 ml&nbsp; 0.2 M CoCl2-6H2O&nbsp; 4.76 g/100 ml<br>2 ml&nbsp; 0.1 M CuCl2-2H2O&nbsp; 1.70 g/100 ml<br>1 ml&nbsp; 0.2 M NiCl2-6H2O&nbsp; 4.76 g/100 m<br>2 ml&nbsp; 0.1 M Na2MoO4-2H2O&nbsp; 2.42 g/100 ml<br>2 ml&nbsp; 0.1 M Na2SeO3&nbsp; 1.73 g/100 ml<br>2 ml&nbsp; 0.1 M H3BO3&nbsp; 0.62 g/100 ml<br><br><br> 

=== Lysis Buffer<br>  ===

50 mM Tris-HCl, pH 8.0 <br>300 mM NaCl <br>10 mM imidazole <br>10% (v/v) glycerol <br>0.2% (v/v) NP-40 <br>0.01% (v/v) 1-thioglycerol or 1 mM beta-ME<br> 

=== Washing Buffer<br>  ===

50 mM Tris-HCl pH 8.0<br>300 mM NaCl<br>'''20''' mM imidazole<br>10% (v/v) glycerol<br>0.01% (v/v) 1-thioglycerol or 1 mM beta-ME<br><br> 

=== Elution Buffer<br>  ===

50 mM Tris-HCl pH 8.0 <br>300 mM NaCl <br>'''150''' mM imidazole <br>10% (v/v) glycerol <br>0.01% (v/v) 1-thioglycerol or 1 mM beta-ME<br><br> 

=== GST-3C Washing Buffer  ===

50 mM Tris-HCl pH 8.0<br>300 mM NaCl<br>10% (v/v) glycerol<br>0.01% (v/v) 1-thioglycerol<br><br> 

=== GST-3C Elution Buffer<br>  ===

50 mM Tris-HCl pH 8.0 <br>300 mM NaCl <br>10 mM reduced glutathione <br>10% (v/v) glycerol <br>0.01% (v/v) 1-thioglycerol or 1 mM beta-ME<br><br> 

<br> 

=== Storage buffer<br>  ===

50 mM Tris-HCl pH 8.0<br>150 mM NaCl<br>10 mM EDTA<br>50% (v/v) glycerol<br>1 mM DTT<br><br> 

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== Protocol 2<br>  ==

=== Expression<br>  ===

Vector: 6XHis rhinovirus 3C protease&nbsp; in a pDB.His.GST expression vector 

Strain: E. coli Rosetta BL21(DE3)plyS, or Nico(21)-pLysN<br> 

Grow in 2YT (5 g NaCl, 16 g tryptone, 10 g yeast extract) or LB containing 50 μg mL−1 kanamycin and 34 μg mL−1 chloramphenincol.<br> 

Inoculate&nbsp; 7 liters of culture with 20 ml/L of an overnight starter culture grown from a freshly transformed single colony<br> 

Grow at 37 °C to OD600 =0.8. <br> 

Lower temperature to 30 °C. <br> 

After approximately 30 min at 30 °C,&nbsp; add IPTG to 0.2 mM. Grow o/n at 30 °C.<br> 

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=== Sample Prep<br>  ===

Spin down cells.<br> 

Resuspend in 50 mL lysis Buffer per liter culture (Buffer A: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM beta-ME, 10% glyerol, 0.1 mM EDTA,&nbsp; 10 mM imidazole). <br> 

Freeze cells with dry ice. 

Lyse cells by thawing&nbsp; in water bucket. 

Discrupt sticky DNA using a homogenizer.<br> 

Spin at&nbsp; 10,000 rpm for 20 min (high speed centrifuge),&nbsp; then 25000 rpm for 20 min (ultracentrifuge), at 4 oC, collect supernatant<br> 

=== Ni Column<br>  ===

Load supernatant into 10 to 20 ml Ni column equilibrated with buffer A. Do not discard Flow through.<br> 

Wash with &gt;5 CVs of Buffer A supplemented with 10 mM imidazole.<br> 

Elute protein with Buffer B (Buffer A + 300 mM imidazole), 2~5 CVs. Collect 3 ml per fraction.<br> 

Check protein&nbsp; in each fraction (10 ul) using Braford reagent (75 ul)<br> 

Combine fractions, set aside 20 ul for SDS-PAGE analysis 

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=== Size-exclusion TSK Gel 3000 SW<br>  ===

50 mM Sodium Phosphate Buffer,&nbsp; pH 7, or Tris-HCl, pH 8.0,&nbsp; 200 mM NaCl,&nbsp; 5 mM beta-ME, 1 mM EDTA and 5% (by vol.) glycerol,<br> 

Collect 3ml per fraction. 

Run SDS gel to see if protein is pure 

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=== Anion/Cation Exchange Chromatography<br>  ===

If purity is not good:<br> 

*Q Sepharose 50 mM Tris-HCl pH 8.8 - 9, &nbsp;5 mM beta-ME. Usea linear NaCl gradient (0 - 1 M NaCl) to elute

*or S-Sepharose fast-flow column. Load crude extract in 50 mM sodium acetate, pH 6.5, 5 mM beta-ME. Usea linear NaCl gradient (0 - 0.5 M NaCl) to elute

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Guideline: 

Buffer pH &gt;PI: protein will bind anion exchange column. 

Buffer pH &lt;PI: protein will bind cation exchange column. 

=== Concentration of enzyme by dialysis<br>  ===

Dialyse purified 3C protein over night at 4 °C with '''Glycerol Concentration Buffer''' (50 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 5 m DTT, 50% glycerol) to 10 mg∕mL 

Aliquot and store at −80 °C.<br><br><br> 

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[[Category:Protocols]]