Dissolution, refolding and ion exchange chromatography experiments on inclusion body precipitates (σ32)

Summary

This experiment describes the solubilization, refolding and ion exchange chromatography of inclusion body precipitation (Σ32). This experiment was derived from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.

Operation method

Dissolution, refolding and ion exchange chromatography experiments on inclusion body precipitates (σ32)

Materials and Instruments

Inclusion Body Precipitation Buffer A Sodium deoxycholate Sodium N-dodecyl sarcosinate
Tissuc-TearorTM homogenizer Dialysis bag POROS 50S cation exchange column SDS-PAGE Electrophoresis unit

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Materials and equipment

Inclusion body precipitation (see Experiment 1, P.147)

^{Tissuc-TearorTM} homogenizer (FisherScientific15-338-55)

Dialysis bag (Spectra/PorTM6; molecular weight cutoff 25000 Da)

POROS 50S cation exchange column (PerSeptive Biosystems)

SDS-PAGE Electrophoresis Unit

Reagents

Buffer A

Buffer A+1mol/L NaCl

Buffer A+50% glycerol

Sodium deoxycholate (DOC) (20% stock solution)

Sodium N-dodecyl sarcosinate (SKL) (20% stock solution)

(For formulations, see "Preparation of Reagents," PP.184~189)

Operating Procedures

Dissolve the inclusion body precipitate with sodium N-dialkylsarcosinate.

1) Add 18 ml of Buffer A and 2 ml of 20% DOC reservoir solution to the inclusion body precipitate (DOC net concentration is 2%). Resuspend the precipitate well with a Tissue-Tearor and leave for at least 10 min at room temperature.
Note: Before washing, the white round portion of the inclusion body precipitate is the inclusion body protein. The brown layer on top is various cellular debris, which is solubilized quite efficiently by 2% DOC. At low temperatures and in the absence of proteins, 2% DOC will form a hydrogel at pH below 8.0. To obtain a gel, increase the pH and temperature.

2) Centrifuge the suspension at 4°C for 10 min at 13,000 r/min and remove the supernatant before discarding (Sample C). To ensure that the precipitate is adequately washed, resuspend the precipitate again as described in step 1. Divide the suspension into two equal parts (tubes #1 and #2) and centrifuge at 4°C, 13,000 r/min for 10 min (a second sample of the supernatant is not required).

Protocol Supplementary Experiment: Prior to the 2nd centrifugation at 13000r/min, 6X50ul of suspension were transferred to Eppendorf tubes individually to determine the amount of SKL required to dissolve the inclusion body precipitate (see p.173).

Note: The 2nd wash of inclusion bodies is to remove cellular debris left in the precipitate plants from the 1st centrifugation. The orange color of the precipitate is due to the use of rifampicin in the cell culture. If rifampicin is not used, the precipitate should be approximately white. If the precipitate is brownish or dark cheese-colored, this is most likely due to the presence of unbroken cells.

3) To the precipitate in tube #1, add 19.7 ml of Buffer A and 0.3 ml of 20% SKL Reservoir (0.3% net SKL concentration). The precipitate should be stirred vigorously to dissolve slowly. The solution should then be allowed to stand for at least 30 min.
Note: 1. By this point, most of the insoluble proteins are denatured, so there is no need to worry about vigorous stirring; 2. SKL is a mild anionic detergent that solubilizes many inclusion body proteins, and renatures the proteins when it is removed from the proteins by dialysis or chromatography SKL is protein bound, so it is important to add a sufficient amount of SKL to titrate the proteins - it appears that at least 1 mg of SKL is required. SKL binds to proteins, so sufficient SKL must be added to titrate the protein - it appears that at least 1 mg SKL/mg of protein is required. When the protein concentration is high, SKL may be required at a concentration less than the critical micelle concentration (CMC; ~0.4%).

Protocol Supplementary Experiments: 2 Precipitation in tubes will be used to test other methods of proteolysis and refolding (see PP,179~181).

4)# Protein suspension solubilized in tube 1, centrifuge at 4°C, 13000r/min for 10 min. collect supernatant (take sample D), discard precipitate (there should not be too much precipitate).

Dialysis removes the detergent to refold the soluble protein.

1) Protein quantification of the dissolved material [Bovine serum albumin (BSA) containing 0.3% SKL must be used for the standard curve]. Dilute with buffer A + 0.3% SKL and adjust the concentration of soluble protein to 1 mg/ml.

2) Dilute the solubilized protein preparation solution 10 times with buffer A so that the final concentration of protein is about 0.1 mg/ml and the final concentration of SKL is about 0.03%.

3) Dialyze 2000 ml of Buffer A against 2000 ml of Lysate Preparation (volume of about 200 ml) for 8 hours at 4°C with thorough agitation. Then change to fresh buffer and repeat.

Additional protocol experiments: Every 4 h, a 150-ul sample is taken and the descaler content is determined by reversed-phase high-performance liquid chromatography (HPLC). This will determine how long it takes to remove SKL by dialysis and how much descaler is present in the sample added to the ion exchange column (see P.176).

Note: When dialyzing a solution containing SKL (or many other detergents), a slow gradient of detergent removal can be left in place, which is a very useful step to allow the protein time to go through the various conformational states so that it can fold correctly, while avoiding undesired hydrophobic aggregation. It appears that aggregation will occur if all SKL, ^σ32 and some other proteins are removed by dialysis. Therefore, the dialysis operation recommended here should not completely remove the SKL, but rather slowly reduce it to 0.01% to 0.02%, and the remaining descaler can be removed in the next step, when ^σ32 is bound to the ion exchange column, washed through the column, and then eluted with a gradient of salt solution.

Ion exchange chromatography

1) Remove the dialyzed protein solution from the dialysis bag and centrifuge for 20 min at 8000 r/min at 4°C to remove all aggregates.

2) The supernatant (Sample J) is loaded onto a POROS 50S cation exchange column for the final stage of separation.

Note: At pH 7.9, the charge of ^σ32 is the sum of -46 and +40; for the titration curve of ^σ32, see Figure 0-2.p.3 in the Introduction section of this book). Since the net charge is -6, ^σ32 can be combined with the positively charged quaternary ammonium group on the POKOS 50Q anion exchange column.

The net charge is -6, so σ32 binds to the positively charged quaternary ammonium group on the POKOS 50Q anion exchange column. Other anion exchange columns such as Q Sepharose Fast Flow, Mono Q, etc. can also be used. It is expected that all residual SKL will bind to POROS 50Q, which is also capable of binding to cation exchange columns due to the large number of positively charged residues. Therefore, it is also possible to purify ^σ32 on negatively charged ion exchange columns such as POROS 50S, S-Sepharose Fast Flow, or Mono S. However, cation exchange columns are preferred over anion exchange columns for the purification of ^σ32 because residual SKL and SKL dissociated from bound proteins will pass through the columns and will not be bound to the columns. In fact, it may even be possible to dispense with dialysis altogether. Simply load the diluted protein sample directly onto the POROS 50S column.

3) Wash the chromatography medium with Buffer A and pour it into a glass column with adapter fittings to make a total volume of 5 ml. Wash the column with Buffer A + 1 mol/L NaCl and equilibrate the column with Buffer A.

4) If dialysis is performed under low salt conditions (as in this experiment), the protein sample can be loaded directly onto the chromatography column at a flow rate of 4 ml/min at room temperature.



5) The column was washed with buffer A for 15 min, followed by a gradient elution (0~lmol/L NaCl/buffer A) at a flow rate of 4 ml/min for 60 min. Absorbance at 260 nm and 280 nm was detected and 4-ml fractions were collected.

6) Analyze the fractions by SDS-PAGE electrophoresis (see Appendix 5) and combine the peak fractions for further analysis. The combined peak fractions can be dialyzed against Buffer A + 50% glycerol, prepared, and stored (as described below). Figure 3-2 shows a typical SDS gel electrophoresis photograph that exemplifies the results obtained in this experiment.

Storage of Purified Proteins

1) Dialyze the combined peak fractions against replacement Buffer A + 50% glycerol overnight at 4°C.

2) Measure _A280nm and follow the method described in Experiment 5 . (PP.167~169) Determine the extinction coefficient and calculate the protein concentration

3) For short term (days to weeks) store at -20°C; at this temperature the sample will not freeze due to the presence of 50% glycerol. Long-term storage should be at -70°C.

Note: This method of storage has several advantages. First, because the water molecules in the dialysis bag pass out of the bag faster than the glycerol enters, the sample can be concentrated by a factor of about three. Second, most enzymes are stable at -70°C for several years under these conditions. A disadvantage of this method is that some enzymes must have the glycerol removed before enzymatic or protein chemistry studies can be performed. If the protein concentration is high enough, it can be diluted until the effect of glycerol is negligible. Otherwise, the sample needs to be dialyzed to remove the glycerol. Another disadvantage is the high cost of reagent grade pure glycerol (~$31/L). A trick to save glycerol is to dialyze first in a tall graduated cylinder without stirring. The water in the dialysis bag quickly leaks out of the bag, which is why it is concentrated by a factor of 3 and floats to the top of the cylinder because it is less dense than the 50% glycerol storage buffer. Carefully decant 10% to 20% of the top layer of buffer to remove most of the water and then equilibrate the buffer in the cylinder. This will effectively replace the buffer without having to use more glycerol.

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