Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from
Escherichia coli (1)
High level expression of recombinant protein in E.coli leads to the formation of highly aggregated proteins referred to as inclusion bodies.
Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The components are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets.
Extraction of recombinant protein using guanidine·HCl
Using the tissue homogenizer, resuspend the washed pellet in guanidine·HCl-containing extraction buffer. Use 1.0 ml buffer per gram wet weight of original cells if the extract will be subjected to gel filtration, and 2 to 4 ml buffer if the extract will be used in protein folding procedures. Perform this step at room temperature.
Extraction buffer
100 mM sodium phosphate/10 mM Tris·HCl, pH 8.0
8 M guanidine·HCl (764 g/liter)
2 mM 2-mercaptoethanol (2-ME).
Store (without 2ME) for at least 1 month at 4◦C
SDS-PAGE of the guranidin extracted recombinant protein
1. Pipet 25 μl sample containing the protein of interest into a 1.5-ml microcentrifuge tube.
2. Add 225 μl cold (0◦ to 4◦C) 100% ethanol to the sample in the tube. The final ethanol concentration is 90% by volume.
3. Mix the sample and ethanol by vortexing. Chill 5 to 10 min at –20◦C or colder (e.g., –80◦C).
4. Microcentrifuge the sample 5 min at maximum speed (∼15,000 × g), 4◦C. Carefully withdraw the supernatant and retain the pellet. The pellet may be difficult to see.
5. Resuspend the pellet in 250 μl cold 90% (v/v) ethanol. Mix thoroughly using a vortex mixer.
6. Microcentrifuge the sample 5 min at maximum speed, 4◦C. Carefully pipet off the supernatant and resuspend the pellet in 25 μl of 1× SDS sample buffer.
7. Heat the sample 3 to 5 min at 90◦ to 100◦C. Load on an SDS-polyacrylamide gel.
Polyhistidine-tagged proteins
Polyhistidine-tagged fusion proteins that form inclusion bodies can be extracted with guanidine·HCl and purified directly by nickel-chelate affinity chromatography.
Extraction
Note: EDTA and DTT react with nickel to form a black precipitate and will ruin the affinity resin. If a reductant is required, use 2 mM 2-mercaptoethanol (2-ME).
A buffer of 100 mM sodium phosphate/10 mM Tris·HCl, pH 8.0 should be used for the extraction with guanidine·HCl. After extraction of the protein, the solution is diluted with buffer to final guanidine·HCl concentration of 4 M to 6 M. The extract is loaded onto the nickel chelation column equilibrated with buffer containing guanidine·HCl and washed with column buffer until no UV absorbance is observed in the elute. The protein may be eluted from the column by either lowering the pH (gradient: pH 8.0 to 3.0) or by imidazole (gradient: 0 to 300 mM).
Note: Heating the solution will also aid protein solubilization; 10 to 15 min at 50◦ to 60◦C is usually a good starting point.