Bacteria engineering bacterium intracellular expression is mainly divided into two forms, one is under the condition of strong promoter of efficient expression, as a result excessive expression of the protein, the protein does not occur without timely and effective folding rules curly, piled up in the form of solid particles in the cell mesenchyme, this is known as inclusions, another is the soluble expression within the stroma, which can occur normally fold, with biological activity. In general, bacteria can separate the inclusion body from the soluble expressed protein by centrifugation as long as the wall is broken normally.
Ultrasonic crushing conditions are generally 300w, 10s/10s, 20 minutes, specific conditions can be determined according to their own conditions.
Preparation of bacteria before ultrasound: after centrifugation, the bacteria were first washed by precipitation with PBS for 2-3 times, and then lysate was added to resuspend the bacteria according to 1/5-1/10 of the volume of the original bacteria liquid. The composition of lysate was 50mmtris-hcl, pH8.0, 2mM EDTA, 100mM NaCl, and lysozyme was added to 100ug/ml, 0.1%Triton x-100. Remember ice bath ultrasound!
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I. how to judge whether the ultrasound is complete? According to experience, there are generally the following aspects:
1. External judgment: the bacterial suspension was turbid before the ultrasound, and became transparent and clear after the ultrasound was complete.
2. Viscosity of the liquid: after ultrasonic, the fungus liquid drops from the gun head without adhesion.
3. High speed centrifugation: high speed centrifugation can be used to detect the degree of ultrasonic breakage (generally 6,000 g 10 min, a little higher than the speed of general centrifugation to collect bacteria). The precipitate is an unfragmented or incomplete thallus.
4. Dyeing: smear the broken fungus liquid, stain with gram crystal violet solution for 0.5 minutes, and examine under the microscope.
(nuclease is added after ultrasound to eliminate the contamination of protein by nucleic acid)
Ii. Some problems needing attention:
1. The protein is expressed in the form of inclusion body, which pursues a high fragmentation rate and requires small cell fragments, while the other protein is expressed in a soluble form, so cell fragments cannot be small. In both cases, different requirements are required but the purpose is the same.
2. If black precipitation occurs during ultrasound, the ultrasonic power is too strong.
3. Too long ultrasonic time and too high power will definitely affect the protein activity.
4. Try to prevent the formation of bubbles.