Abstract: The separation and purification of proteins is relatively complicated. Proteins extracted from cells or proteins obtained by precipitation, gradient centrifugation, and salting out of solutions containing proteins often contain impurities. To remove these impurities, at the same time, the protein must be maintained The biological activities, such as the catalytic activity of enzymes, need to develop corresponding strategies and adopt different methods according to different proteins.
1. General objectives and methods of purification
First, natural-sourced or recombinantly expressed proteins undergo some crude extraction steps (eg, homogenization, centrifugation, ammonium sulfate precipitation, etc.) to become stable samples that can be used for chromatographic separation.
Then capture chromatography (capturechromatography), the main goal is to concentrate and remove a large number of easily removed impurities, this step is most concerned about the flow rate and capacity, often using high-load, fast-flow gel.
The concentrated partially purified sample is subjected to intermediate chromatography (intermediatechromatography), the purpose is to remove impurities that are more difficult to remove, the most important thing in this step is resolution, often using high-resolution fine-grain gel.
Finally, in order to obtain the final product that meets the requirements, the remaining impurities and the polymer or degraded fragments of the target protein are removed, and polishing chromatography is carried out. Gel filtration chromatography with high-resolution gel filtration gel is often used.
2. Preparation before purification
(1) Sample stability test
a. Determine the stability of the sample at pH 2-9;
b. Determine the stability of the sample in 0-4mol / LNaCl and 0-2mol / L ammonium sulfate;
c. Determine the stability of the sample in 0-50% ethanol and methanol;
d. Determine the stability of the sample at 4-40 ℃;
e. Leave at room temperature overnight to determine the stability to proteolytic enzymes.
(2) Sample pretreatment
a. Remove the particulate matter in the sample (0.45-0.22 micron membrane filtration or 10000g centrifugation for 15 minutes)
b. Remove the lipids in the sample (centrifugation at 10,000g for 15 minutes or organic solvent extraction)
c. Remove the nucleic acid in the sample (add nuclease to digest or precipitate nucleic acid)
d. Inhibit proteolytic enzymes in the sample (add protease inhibitors, rapid first step separation at low temperature or express recombinant protein in protease-deficient hosts)
(3) Storage conditions of samples:
Short-term storage (less than 24 hours)
a. Avoid approaching or exceeding the stability limit of the sample to prevent protein denaturation or precipitation
b. Refrigerate in a closed container
Long-term storage (several days)
a, b, ibid
c. Add appropriate bacteriostatic agent
Long-term storage
a, ibid
b. Frozen or best freeze-dried (vacuum freeze-dried).
3. Establishment of evaluation methods for each purification step
a. Determination of target protein content
Enzyme activity measurement, biological activity measurement, radioimmunoassay, enzyme-linked immunoassay, immunoelectrophoresis, fluorescence.
b. Determination of total protein content
Ultraviolet absorption method, Lowry method, Bradford dye binding method (Coomassie Brilliant G-250), etc.
c. Sample complexity detection
HPLC (ion exchange, gel filtration, reverse phase), SDS-PAGE, isoelectric focusing, capillary electrophoresis (CE).
4. Source of protein
(1) Natural protein: The target protein in natural products generally has little content and extremely complex components. Multiple steps must be used in the separation process to remove various impurities, and the activity of proteolytic enzymes should be reduced throughout the process.
(2) Recombinant protein: Recombinant protein has higher target protein abundance. There are three main expression localizations of recombinant proteins:
a. Cytoplasm: In this case, the cell structure must be destroyed to obtain the target protein, accompanied by the release of a large number of nucleic acids and other thousands of host proteins; under the conditions of high expression, inclusion bodies are generally formed. It contains over-expressed protein of interest and is easily separated by high-speed centrifugation, but the dissolution and renaturation of inclusion bodies is more complicated.
b. Peripheral substance: The expressed protein exists between the inner and outer membranes of bacteria, so that the target protein can be less affected by proteases and reduce the contamination of the host protein.
c. Secretion to the culture medium: this expression generally has a low protein concentration, mainly due to contamination in the culture medium.
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