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  • Purification of a Fusion Protein generated by The pMAL Protein Fusion and Purification System (E8200)

    Introduction

    Two protocols for purification of a fusion protein from a 1 liter culture are detailed below. Method I is designed for purification of a soluble fusion protein expressed in the cytoplasm from a pMAL-c5 vector. However, the crude extract prepared by this method also contains periplasmic proteins, so it can be used to prepare secreted fusion proteins expressed from the pMAL-p5X vector as well. Method II is designed for purification of a secreted fusion protein expressed from the pMAL-p5X vector (13). It results in a periplasmic fraction that contains many fewer E. coli proteins than the total cell crude extract prepared by method I, but the procedure is more difficult to scale up.

    Protocols

    1. Method I – Total cell extract 

      1. Inoculate 1 liter rich broth + glucose & ampicillin with 10 ml of an overnight culture of cells containing the fusion plasmid. 

      Glucose is necessary in the growth medium to repress the maltose genes on the chromosome of the E. coli host, one of which is an amylase which can degrade the amylose on the affinity resin. 

      2. Grow to 2 x 108 cells/ml (A600 ~0.5). Add IPTG to a final concentration of 0.3 mM, e.g. 72 mg or 3 ml of a 0.1 M stock in H2O. Incubate the cells at 37°C for 2 hours. 

      The period of time and the temperature to use during expression depends on several factors (stability of the protein, host strain, etc.), and variations can be tried to find optimum conditions. 

      3. Harvest the cells by centrifugation at 4000 x g for 20 minutes and discard the supernatant. Resuspend the cells in 25 ml Column Buffer. 

      For many unstable proteins, most of the degradation happens during harvest and cell breakage. Therefore, it is best to harvest the cells quickly and keep them chilled. 25 ml of Column Buffer is based on the expectation of about 4-5 grams cells/liter, i.e. 5 ml for every gram of cells (wet weight). 

      The EDTA in the lysis buffer is to help inhibit proteases that have a Ca++ cofactor. Addition of PMSF (phenyl methylsulfonyl fluoride) and/or other protease inhibitors may help in some cases. 

      DTT or β–mercaptoethanol can be included to prevent oxidative damage to the fusion protein and interchain disulfide bond formation upon lysis (disulfide bonds usually do not form intracellularly in E. coli). 

      4. Freeze sample in a dry ice-ethanol bath or overnight at -20°C; -20°C is more effective than -70°C, but takes longer. Thaw in cold water. 

      This is a good place to stop the protocol overnight – the frozen cells can be stored for a week or more at -20°C, depending on the particular fusion. 

      5. Place sample in an ice-water bath and sonicate in short pulses of 15 seconds or less. Monitor the release of protein using the Bradford assay (14), adding 5 µl of the sonicate to 1 ml Bradford reagent and mixing. Continue sonication until the released protein reaches a maximum (usually about 2 minutes sonication time). 

      6. Centrifuge at 20,000 x g for 20 minutes. Save the supernatant (crude extract). Dilute the crude extract 1:6 with Column Buffer. 

      This is another good place to stop the protocol – the crude extract can be stored for a week or more at -20°C, depending on the particular fusion. It is usually more convenient to store the crude extract before diluting with column buffer, then to dilute it after removal from storage and thawing. 

      7. Pour the amylose resin in a 2.5 x 10 cm column. Wash the column with 5 column volumes of Column Buffer. 

      The amount of resin depends on the amount of fusion protein produced. The resin binds about 6-8 mg/ml bed volume, so a column of about 15 ml should be sufficient for a yield of up to 100 mg fusion protein/liter culture. 

      8. Load the diluted crude extract at a flow rate of no more than [50 x (diameter of column in cm)2 ml/hour. This is a maximum of 5 ml/minute for a 2.5 cm column. 

      9. Wash with 12 column volumes of Column Buffer at a rate of no more than [100 x (diameter of column in cm)2 ml/hour. This is a maximum of 10 ml/minute for a 2.5 cm column. 

      The column can be washed overnight, if it has a safety loop to prevent it from running dry. In this case, it is better to restart the column with elution buffer (step 10), rather than continuing the wash. Avoid loading the column overnight.
       
      10. Elute the fusion protein with Column Buffer + 10 mM maltose. Collect 10 to 20 fractions of 3 ml each (fraction size = 1/5th column volume). The fusion protein usually starts to elute within the first 5 fractions, and should be easily detected by UV absorbance at 280 nm or the Bradford protein assay (14). 

      11. Pool the protein-containing fractions. If necessary, concentrate to about 1 mg/ml in an Amicon Centricon or Centriprep concentrator, an Amicon stirred-cell concentrator (Millipore), or the equivalent.
    2. Method II – Periplasmic extract 

      1. Inoculate 1 liter rich broth + glucose & ampicillin with 10 ml of an overnight culture of cells containing the fusion plasmid. 

      2. Grow to 2 to 4 x 108 cells/ml (A600 ~0.5). Add IPTG to a final concentration of 0.3 mM, e.g. 72 mg/l or 3 ml of a 0.1 M stock in H2O. Incubate the cells at 37°C for 2 hours. 

      The period of time and the temperature to use during expression depends on several factors (stability of the protein, host strain, etc.), and variations can be tried to find optimum conditions (see Pilot Experiment). In addition, partial induction of exported proteins may lead to higher yields, since protein export in E. coli may not be able to keep up with full level Ptac expression. 

      3. Harvest the cells by centrifugation at 4000 x g for 20 minutes and discard the supernatant. Resuspend the cells in 400 ml 30 mM Tris-HCl, 20% sucrose, pH 8.0 (80 ml for each gram of cells wet weight). Add EDTA to 1 mM and incubate for 5–10 minutes at room temperature with shaking or stirring. 

      4. Centrifuge at 8000 x g for 20 minutes at 4°C, remove all the supernatant, and resuspend the pellet in 400 ml of ice-cold 5 mM MgSO4. Shake or stir for 10 minutes in an ice bath. 

      5. Centrifuge at 8000 x g for 20 minutes at 4°C. The supernatant is the cold osmotic shock fluid. 

      6. Add 8 ml of 1 M Tris-HCl, pH 7.4 to the osmotic shock fluid. 

      7. Continue from Method I, step 7. 

      Regenerating the Amylose Resin Column 

      The resin may be reused three to five times when regenerated with the following sequence of washes: 

      Water: 3 column volumes
      0.1% SDS: 3 column volumes
      Water: 1 column volumes
      Column Buffer: 3 column volumes

      Please note that although the column can be washed at 4°C, 0.1% SDS will eventually precipitate at that temperature. It is therefore recommended that the SDS solution be stored at room temperature until needed, and rinsed out of the column promptly. Upon repeated use, trace amounts of amylase in the E. coli extract decrease the binding capacity of the column. It is recommended that the column be washed promptly after each use.