Experiments on the fragmentation of Escherichia coli cells and preparation of inclusion bodies
Experiments on the fragmentation of Escherichia coli cells and preparation of inclusion bodies
This experiment describes the fragmentation of E. coli cells and the preparation of inclusion bodies. This experiment is from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.
Operation method
Experiments on the fragmentation of Escherichia coli cells and preparation of inclusion bodies
Materials and Instruments
E. coli cell line overexpressing σ32 Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Chloramphenicol Ampicillin Isopropylthio-β-D-galactoside Rifampicin LB medium SDS Sample Buffer Sodium deoxycholate
Oak Ridge Centrifuge Tubes Graduated Polypropylene Conical Centrifuge Tubes Tissue-TearorTM Homogenizer Ultrasonic Breaker
E. coli cell lines overexpressing σ32 [BL21(DE3)/pLysS, pLHN16
Chloramphenicol
Ampicillin
Isopropylthio-beta-D-galactoside (IPTG)
Rifampicin
Oak Ridge Centrifuge Tubes (40-ml)
Graduated polypropylene conical centrifuge tubes (50-ml)
Tissue-TearorTM Screeder (Fisher Scientific15-338-55)
Ultrasonic Breaker
Reagents
LB medium
SDS Sample Buffer (2X)
Sodium deoxycholate (DOC) (20% stock solution)
(For formulations, see "Reagent Preparation", PP.184~189)
Operating Procedures
Preparation of raw materials
The E. coli cell line used in this unit [overexpression plasmid for σ32 is pLHN16, and the host bacterium is BL21(DE3)/pLysS] was constructed according to the method described by Nguyen et al. (1993) [see the pETTM System Manual published by Nuvagen (Novagen, Inc. 1995) for the application of the pETll expression system]. The pETll expression system was constructed according to the method described by Nguyen et al.
1) E. coli was incubated at 37°C in shake flasks of 2000-ml volume, each containing 500 ml of LB medium, under a selection pressure of chloramphenicol (25ug/ml)/cyanobenzylpenicillin (100ug/ml) until the A550nm reaches 0.9.
2) Add IPTG to a final concentration of 1 mmol/L to induce T7RNA polymerase expression. After 0.5 h of induction, rifampicin was added to a final concentration of 150ug/ml to maximize overexpression.
3) End the culture 3.5 hours after rifampicin addition and centrifuge the cells at 4°C for 30 min at 8000r/min. Collect the cells by centrifugation at 8000r/min for 30 min at 4°C. Resuspend the cell precipitate from each liter of culture in 30 ml of LB medium and centrifuge the cells in 40-ml Oak Ridge tubes at 13000r/min for 10 min at 4°C. Freeze the cells on dry ice and store at -70°C for later use.
Note: Although the cells used in this module are frozen, for unknown proteins it is much safer to prepare them in fresh cells, especially if refolding is required. Cells should preferably be frozen for no more than 5d (days).
Sampling for monitoring purification steps
To monitor the progress and effectiveness of purification, it is necessary to perform the following manipulation steps for each fraction at each stage of purification.
1) Measurement of volume.
NOTE: A very convenient way to prepare a set of calibrated tubes for quick measurement of the volume of the solution is to carefully add H2O to the various sizes of tubes used, e.g., Eppentbrf tubes, Oak Ridge centrifuge tubes, etc., and mark the horizontal line corresponding to each volume. Graduated conical centrifuge tubes can also be conveniently used for this purpose.
2) Add a 36-ul sample to 84ul of SDS Sample Buffer (2X), heat in a water bath at 70-90°C for 2-5 min, and store at 4°C.
3) A portion of the sample (150ul) is retained for protein determination and immuno-quantitation. Record the above information on the Purification Record Sheet and use it to prepare a Purification Summary Sheet. Care must be taken to retain a representative sample. This means that the whole fraction should be really well mixed before sampling. This is especially important for those fractions that contain precipitated or resuspended insoluble material. For convenience, when setting aside samples for SDS gel electrophoresis analysis and protein quantification, a table can be created listing Samples A through J, their associated descriptions and volumes, and the two chromatographic columns to be examined. In this unit, you will also be instructed to take samples at different stages of purification (as described in the respective experiments), which will be used in complementary experiments to the purification protocol, as described in the last section of the unit.
Ultrasonic cell fragmentation
1) Take 3 g of E. coli cells [BL21(DE3)/PLysS, pLHN16] and thaw at room temperature. Cells were briefly homogenized with a tissue breaker and resuspended in 20 ml of buffer A (in a 40-ml Oak Ridge centrifuge tube).
Note: When purifying σ32, no reducing agent is added to Buffer A, as σ32 does not contain cysteine residues. Typically, all buffers are supplemented with (0.1-0.5 mmol/L dithiothreitol to prevent protein oxidation.
2) Using a large sonication head, set 8 cycles and 50% time interval, sonicate for 90s in an ice bath.
Note: BL21 DE3 cells carry pLysS that expresses the T7 lysozyme callus, however, the lysozyme cannot attack the cell wall from the inside, so the gene is not lethal. When the cells are frozen and thawed, the lysozyme gets close to the cell wall and lyses the cell. In some cases, this is enough to rupture the cell, but the viscosity problem due to the release of DNA must be solved. One approach is to digest with a fairly large amount of DNAase I, but this can be expensive and adds another protein to the mixture, which can cause further problems (e.g., if DNA-binding proteins are being purified). For this reason, to ensure that the cells are completely pulverized, we use sonication. This cuts the DNA to a large extent and makes precipitation of inclusion bodies easier.
3) Add DOC to a final concentration of 0.2% (i.e., add about 240ul of 20% DOC reservoir). Mix well and let stand for 10 min.
Note: 1. 0.2% DOC is used to help release small amounts of insoluble proteins and, at higher concentrations (2.0%), components of the cell membrane (see Experiment 2). DOC dissolves quite slowly, so the reservoir should be made up to 1 day in advance. 2. If you want to isolate only the inclusion bodies, you may want to use the DOC reservoir for a few days. If only inclusion bodies are to be separated and no further separation of soluble proteins in the supernatant is desired (Sample B below), DOC can be added at this step to a final concentration of 2% and the second DOC wash described in Experiment 2 can be omitted.
4) Take the first sample (Sample A), as described above in p. 146).
Preparation of inclusion bodies and soluble extracts
1) To precipitate the inclusion bodies, the cell lysates were centrifuged for 10 min at 13,000 r/min, 4°C with a small rotary head (SorvallSS-34 or Beckman JA20).
2) Gently decant the supernatant (Sample B) and keep on ice for PEI precipitation and immunoaffinity chromatography (IAC) purification of the core RNA polymerase-P2 complex (see Experiment 3). For lysis and reconstitution of overexpression in inclusion bodies, see Experiment 2.