The purpose of this section is to provide an abbreviated set of working procedures for how to use a series of experiments similar to those described in this unit to quickly obtain an initial purification protocol for a new protein overexpressed in bacteria. This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.
Operation method
New purification experiments of bacterial overexpression products (without disulfide bonds) Move operating procedure For more product details, please visit Aladdin Scientific website.
Day 1
The purpose of today is to test the induction conditions for T7 RNA polymerase expression, determine its distribution in the supernatant/precipitate and make a preliminary assessment for the isolation and purification steps of the next day.
1) Start by taking a 50-ml portion of an E. coli cell culture that has been incubated overnight at low density.
2) Induce T7 RNA polymerase expression with 0.8 mmol/L IPTG at A600nm of 0.3~0.7 and split the culture into two.
3) After 30 min, rifampicin was added to one culture to a final concentration of 150 pg/ml.
4) During the induction process, samples were taken at each time point for SDSPAGE analysis: 1 ml of culture was centrifuged, and 100ul of 2xSDS sample buffer was added to each unit of A600nm to dissolve the precipitate, and the induction time was traced for 2~4 hours.
5) After taking the last sample, combine the cultures and break up the cells by sonication/DOC (see P.146), which should answer the question of whether the protein is soluble or in the precipitate.
6) If the protein is present in the precipitate, it can be initially screened by SKL lysis (see pp.148-149 and p.173). If the protein is present in the supernatant, try screening the supernatant for PEI precipitation/elution conditions (see PP.153~154 and pp.l74~175). After completing these experiments, dispose of everything in the wastebasket and prepare for the Day 2
Prepare a larger volume (1L) of culture.
Day 2
The purpose of today's procedure is to quickly go through the first steps of the protein purification protocol, and to screen the later steps of the column chromatography procedure using 1 liter of freshly prepared culture under the optimal conditions determined on day 1.
1) If the protein is in the soluble fraction, it can be treated by PEI precipitation/elution (PP.153-154) and then screened for binding conditions to different chromatographic media. Generally, it is recommended to try ion exchangers (Q and/or S) and hydrophobic interaction media (phenyl-Sepharose) first. The binding conditions are screened first, and then the elution conditions are screened for optimal binding conditions. Proteins are adsorbed onto the chromatographic medium in a batch mode, and then loaded onto the column and eluted.
2) If proteins are in the precipitation fractions, they may be divided into several portions and solubilized at several protein concentrations at the originally specified SKL levels. Dialyze overnight. Folding experiments with guanidine hydrochloride (GuHCl) or urea can also be tried on several fractions of the precipitate. If the precipitate is not as pure as desired, try washing the precipitate with other detergents (e.g., Triton X-100, Tween-20, Brij-35) or low concentrations of urea or guanidine hydrochloride. Wait until tomorrow and analyze by gel filtration chromatography to determine the amount of monomeric protein as a function of protein concentration and other parameters. Prepare another culture of 1 liter or more.
Day 3
The purpose of today is to go through the main protein purification steps and further test the chromatography steps. Start the activation test and look for electrophoretic isoforms and/or co-migrating impurities using bi-directional gel electrophoresis.
1) If the protein is soluble, the material should be fairly pure, and it is probably time to audit the soluble aggregates and modified proteins and try the activity assay.
2) If the protein is in the precipitate, some sort of chromatography may also be necessary to remove residual detergent.