Defunct: pET29 S100 expression & purification

To recombinantly express and purify S100 proteins from bacteria.

 

• pET29b(+) vector.  This is a T7-based expression vector that uses a kanamycin resistance marker.  Kanamycin is a better choice than ampicillin for expression as it does not break down as the pH drops during culture saturation, forcing the bacteria to maintain the expression plasmid.
• Rosetta BL21(DE3) pLysS cells.  These are competent expression cells that have been modified to maximize protein yield.  The cells lack the Ion protease and the ompT outer membrane protease genes to prevent protein degradation.  They are designed for induction using T7 promoters and the lac gene (e.g. with IPTG).  They have also been manipulated to allow transformation with the desired vector by simple heat shock.  Finally, they possess the Rosetta plasmid, which encodes tRNAs for a variety of low frequency E. coli codons.
• Luria broth. 10 g tryptone, 10 g NaCl, 5 g yeast extract.  Bring to 1 L with ddH2O and autoclave.
• 1,000x kanamycin stock. 30 mg/mL in ddH2O.  Filter sterilize with 22 um filter.  Selects for expression plasmid.
• 1,000x chloramphenicol stock.  34 mg/mL in EtOH.  Selects for pLysS plasmid.
• 1,000x IPTG stock.  0.238 g/mL (1 M).  Keep at 4C.  Induces protein expression.
• 50% glucose (sterile).  Obtain from media kitchen.

• Transform/streak out a glycerol stock of the relevant clone onto an LB kanamycin/chloramphenicol plate.
• Inoculate a 10 mL (LB + kan+cam) culture from a single colony.  Place at 37 °C, shaking @ 220-250 rpm.  Grow overnight.
• Inoculate a 1.5 L (LB + kan + cam) culture with the overnight culture.  Place at 37 °C, shaking @ 220-250 rpm.
• Follow growth by OD600.  Aim for logarithmic growth and an OD600 of 0.8. as measured using a plastic cuvette in the NanoDrop.  (Note: measure the OD600 using the “Cell Cultures” protocol, not the absorbance protocol.  Because of how the NanoDrop processes the raw data, the cell culture and absorbance values are radically different). An OD600 of 0.8 is usually achieved within ~3.5 hr.
• Induce growth by adding 1.5 mL 1M IPTG (1 mM) and 7.5 mL 50% glucose (0.2%).  Drop the growth temperature to 16 °C.
• Allow cultures to grow overnight.
• Spin cultures down for 10 min @ 8,000 rpm and place centrifuge bottles in -20 °C freezer. Leave them in the freezer for ~0.5 hr to overnight.  Tip: leave the bottles on their side as it makes the pellets much easier to pop out after freezing.
• Use a stiff metal spatula to pop the pellets out of the bottles into a 50 mL conical labeled with the protein name, the pellet mass, your name, and the date. Tip: as the pellets thaw they are harder to remove from the bottle.  Pull the bottles out two at a time, rather than all at once, to make it easier to pop the pellets.
• Move onto the lysis step. Otherwise, place the conical with the pellets into the –20 °C freezer.  The pellets will keep for ~1 month.

Component	Molecular weight	Mass
25 mM Tris	121.14 g/mol	0.61 g
300 mM NaCl	58.44 g/mol	3.51 g
1 mM DTT	154.25 g/mol	0.03 g

Bring pH to 7.4 using HCl

 

Component	Molecular weight	Mass
25 mM Tris	121.14 g/mol	6.06 g
100 mM NaCl	58.44 g/mol	11.69 g
1 mM DTT	154.25 g/mol	0.31 g
30% ammonium sulfate	n/a	391.9 g

Bring pH to 7.4 using HCl

 

Use 2X HIC buffer to make 1 L of 1X HIC buffer A and 1 L of 1X HIC buffer B.

• Buffer A: add 500 mL 2X HIC buffer, 500 mL ddH2O, and 2 mL 1 M CaCl2 stock.
• Buffer B: add 500 mL 2X HIC buffer, 500 mL ddH2O, and 10 mL 500 mM EDTA stock.
• Filter buffer A and B at 0.22 um using the vacuum filtration system.
• Place buffer A and B at 4 °C

Protocol