Performing a Cell Lysis of Recombinant Histidine-Tagged Proteins

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

For optimal conditions for growth, induction, and cell lysis of recombinant histidine-tagged proteins, please refer to established procedures. The following is a general procedure for cell lysis and sample preparation from bacterial cultures. Other established procedures may also work.

This procedure works well with the majority of the purification protocols included in this chapter. However, some modifications of the procedures are noted where relevant.

  1. Harvest cells from the culture by centrifugation at 7000 to 8000 × g for 10 min or at 1000 to 1500 × g for 30 min at 4°C.
  2. Discard the supernatant. Place the bacterial pellet on ice.
  3. Dilute the cell paste (bacterial pellet) by adding 5 to 10 ml of binding buffer for each gram of cell paste.
  4. Enzymatic lysis: Add 0.2 mg/ml lysozyme, 20 µg/ml DNase, 1 mM MgCl2, 1 mM Pefabloc™ SC or phenylmethylsulfonyl fluoride (PMSF) (final concentrations). Stir for 30 min at room temperature or 4°C, depending on the sensitivity of the target protein.
  5. Mechanical lysis:
    Sonication on ice, approximately 10 min.
    or

    Homogenization with a French press or other homogenizer
    or
    Freeze/thaw, repeated at least five times.

Mechanical lysis time may have to be extended to obtain an optimized lysate for sample loading to avoid problems with back pressure. This is important when direct loading of unclarified, crude sample is performed (using HisTrap FF crude, HiTrap TALON crude, or HisTrap Excel columns). Different proteins have different sensitivity to cell lysis, and caution should be exercised to avoid heating and frothing of the sample.

  1. Measure and adjust pH if needed.

Do not use strong bases or acids for pH adjustment, as this may increase the risk of precipitation.

If the sonicated or homogenized unclarified cell lysate is frozen before use, precipitation and aggregation may increase. New mechanical lysis of the lysate can then prevent increased back-pressure problems when loading on the column.


Sample preparation

If the sample is prepared in a buffer other than the binding buffer, adjust the sample to the composition and pH of the binding buffer by adding buffer and additives from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.

Pass the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before applying it to the column. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging the column; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.

Note: Filtration is NOT necessary when using HisTrap™ FF crude, His GraviTrap™, His MultiTrap™ HP, His MultiTrap™ FF, HisTrap™ Excel, or HiTrap™ TALON® crude.

If the recombinant histidine-tagged protein is expressed as inclusion bodies, the inclusion bodies are solubilized using 6 M Gua-HCl or 8 M urea, and the chosen denaturant must be present in all buffers during chromatography. Advice for working with inclusion bodies can be found in Chapter 10 (Handling inclusion bodies) and in the troubleshooting section.

 

Materials

     
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