Bacterial Protein Expression and Refolding Protocol

Bacterial Protein Refolding protocol

Outline:
1.   Production of bacterial inclusion bodies
2.   Re-folding of protein
3.   Purification
4.   Chemical and enzymatic modifications
5.   List of Buffers

Production of bacterial inclusion bodies

1.   Create starter culture of 3mL, grow overnight, alternatively use 1mL of frozen glycerol stock and grow overnight in 250mL LB.
2.   Innoculate 4-9 liters LB with 250mL starter culture, grow at 37.
3.   Prepare 500mM-1M IPTG solution (in PBS).
4.   Measure absorbance every 30 minutes after 2 hours and grow to OD600 of between 0.8-1.2.
5.   Induce with 1:1000 dilution of IPTG stock (ie 1mL/L culture).
6.   Induce for 2 hours (this step may be optimized for each protein).
7.   Harvest bacteria by centrifugation, 4000RPM for 10 minutes is adequate. Pelleted bacteria may be stored at 4 C for up to a week. Pelletes may also be frozen if cells will be lysed using sonication, not microfluidizing.
8.   Re-suspend cells in lysis buffer, the smaller the volume the better.
9.   Add 200mL lysozyme (stock 25mg/mL) and 250mL DNAseI (stock 2mg/mL) to every liter of bacteria grown.
10.   Let sit on ice
11.   Sonication protocol

Re-folding of protein
   There are many different protocols for protein refolding that can be utilized. The most commonly used procedure is outlined below, but basically most methods rely on slowly renaturing proteins from Urea or Guanidine in a TEA buffer with oxidized and reduced glutathione to aid in creation of disulfide bonds.

1.   Estimate concentrations of protein/s, approximately 30-40mg protein can be used per 100mL refolding reaction, larger volumes provide more benefit.
2.   For every 100mL refold buffer add: 1mL 100mM PMSF (in isopropanol), 50uL pepstatin, 50uL leupeptin. Keep buffer in plastic container!!
3.   For every 100mL add 30.63mg oxidized glutathione (0.5mM) and 153.65mg reduced glutathione (5mM)
4.   Stir reaction mix in cold room on a slow setting.
5.   Divide up protein to be refolded into about 5 tubes.
6.   Add 250uL of injection buffer to each tube.
7.   Add contents of one tube dropwise into refolding buffer.
8.   Let incubate for about 2 hours and repeat with next tube.
9.   After last tube, let reaction incubate overnight or 8 hours
10.   Centrifuge sample at 4000 RPM for 10 minutes to remove precipitated protein.
11.   Repeat
12.   Concentrate sample using AMICON stirred cell apparatus down to 1mL per mg of original protein used (ie 30mL for 30mg protein).
13.   Use an AMICON filter with a pore size smaller than protein size. Generally for most protein samples a YM-10 filter works fine.
14.   Pre-soak the membrane shiny side down in water for 1 hour.
15.   Assemble the apparatus with the shiny side up.
16.   Fill chamber and concentrate at 50psi.

Column Purification of Proteins

17.   Dialyze sample to Ni or Co resin binding buffer if those are to be used, or size exclusion buffer. Use at least one buffer exchange to insure at least a 10,000 fold dialysis.
18.   Purify sample using manufacturers protocol for Ni or Co resins.
19.   Solubility of eluted protein varies, and thus must be determine using small aliquots.
20.   Standard PBS may cause precipitation, thus a Tris containing buffer is preferred. A buffer containing 20mM Tris-HCl and 50mM NaCl pH 8.0 is usually used.
21.   Dialyze sample to buffer used in size-exclusion buffer (see above). This ensures that no precipitation occurs in column as well as removes imidazole if a Ni or Co resin column is used.
22.   Filter sample with 0.45 m filter and separate by size-exclusion chromatography.
23.   S-100 and G-200 resins may be used for samples up to 100 and 200 kDa respectively.
24.   Flow rate should be ≤0.5mL/min. The smaller the column, the lower the flow rate that should be used.