Protein expression system construction
Protein expression system construction
Summary
E. coli protein expression is a commonly used protein expression system. E. coli is a common bacterium, which has been widely used in the field of bioengineering. By inserting target genes into E. coli expression vectors and utilizing E. coli metabolic pathways and physiological features, target proteins are efficiently expressed in E. coli.
Operation method
Escherichia coli protein expression
Materials and Instruments
Receptor cell BL21(DE3), plasmid (with target gene), LB liquid medium, LB solid medium, Kanamycin/Ampicillin, IPTG, -80 ℃ ultra-low temperature refrigerator, ultra-clean workbench, water bath, icemaker, thermostatic oscillating incubator, autoclave, coated beads, culture flask, pipette gun UV spectrophotometer.
Move
I. Transformation of receptor cells BL21(DE3)
1, take out the BL21(DE3) receptor cells from -80 ℃ ultra-low temperature refrigerator and put them on ice to melt;
2, take 1 μL of plasmid (including the target gene) and add it to 100 μL of BL21 (DE3) receptor cells in the ultra-clean bench, and gently blow and mix with a pipette gun (this step must be mixed gently, to avoid damage to the cells);
3、After 30 min of ice bath, the receptor cells were heat-excited at 42 ℃ for 90 s and then ice bath for 2 min;
4, add 900 μL LB liquid medium (without antibiotic Kanamycin/Ampicillin) to the receptor cells, and put them in a constant temperature incubator at 37 ℃ and 220 rpm for 1 h.
Monoclonal culture
1. Put the LB solid medium sterilized by autoclave into the ultra-clean bench and add antibiotics (50 μg/mL Kanamycin/Ampicillin) when it is not hot. Take a disposable sterile petri dish, pour the medium slowly into the center of the sterile petri dish so that it spreads all over the bottom of the sterile petri dish, and wait until it cools and solidifies;
2. Pour in about 10 coated beads, add 100 μL of transformed sensory cell solution to the petri dish, spread the sensory cell solution evenly on the petri dish, put it in a 37 ℃ constant temperature oscillation incubator for 5 min, let it dry and then inverted to culture overnight;
3, the next day, pick the monoclonal colonies to 10 mL of sterile LB liquid medium (containing antibiotics 50 μg/mL Kanamycin/Ampicillin), placed in a constant temperature oscillation incubator at 37 ℃, 220 rpm overnight culture.
Expanded culture
1. Add 10 mL of overnight culture medium into 1L of sterile LB liquid medium (containing 50 μg/mL Kanamycin/Ampicillin) (the amount of culture can be selected according to this ratio), and place it in a constant temperature oscillation incubator at 37 ℃ and 220 rpm;
2. After 3 hours of incubation, take out 1 mL of culture solution from the culture bottle and measure the OD value by UV spectrophotometer to detect the density of the strain. When OD600 = 0.6 - 0.8, lower the temperature of the shaker to 20 ℃, add 1 mM IPTG to the bacterial liquid for induction, and then centrifuge to collect the bacterial body after 10 h. (The temperature, time and concentration of IPTG can be adjusted according to the different expression proteins), and then store the bacterial body precipitation after centrifugation at -80 ℃.
Caveat
1、Receptor cells should be stored at -80 ℃, and should not be repeatedly frozen or thawed and placed for too long, or reduce their transformation efficiency.
2、During the experimental operation, aseptic operation should be ensured to avoid contamination by stray bacteria.
3， in order to prevent the monoclonal colonies are too dense, can be diluted with different gradient of the receptor cell transformation solution for coating plate.
4， the strain before the expansion of culture is best not to activate too long, it is easy to over the logarithmic growth period.
5、When determining the OD value of the spectrophotometer, use LB liquid medium for calibration, not water, otherwise the error is large.
6， IPTG induced by the temperature, time, IPTG concentration check the literature, you can also do their own pre-test to find out. Because the size and solubility of proteins are not the same, so the experimental conditions for each protein will be different.
7， save the experimental materials of each step, if there is no pure protein, it can be convenient to detect and know which step is wrong, so that it is convenient to improve next time.
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