Editing HRV 3C Expression and Purification (section)

== 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> 

<br> 

=== 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 

<br> 

=== 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 

<br> 

=== 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

<br> 

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> 

<br> 

[[Category:Protocols]]