General objectives and methods for the purification of modified peptides
First, naturally derived or recombinantly expressed proteins are subjected to some crude steps (eg, homogenization, centrifugation, ammonium sulfate precipitation, etc.) to become stable samples that can be used for chromatographic separation.
Capture chromatography is then performed with the primary goal of concentrating and removing large amounts of easily removed impurities. This step is most concerned with flow rate and loading, often using high loading, fast flow gels.
The concentrated, partially purified sample is subjected to intermediate chromatography to remove impurities that are difficult to remove. This step is most concerned with resolution, often using high-resolution fine-grain gels.
Finally, in order to obtain a final product that meets the requirements, the remaining impurities and the polymer or degradation fragments of the target protein are removed, and polishing chromatography is carried out, and a gel filtration gel with high resolution is often used for gel filtration chromatography.
2. Preparation of modified peptides 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 0-4 mol/L NaCl and 0-2 mol/L ammonium sulfate;
c, determining the stability of the sample in 0-50% ethanol, methanol;
d, determining the stability of the sample at 4-40 ° C;
e. Allow to stand overnight at room temperature to determine the stability to proteolytic enzymes.
(2) Sample pretreatment
a, remove the particles in the sample (0.45-0.22 micron membrane filtration or 10000 g centrifugation for 15 minutes)
b, remove the lipids in the sample (1500 g centrifugation for 15 minutes or organic solvent extraction)
c, remove the nucleic acid in the sample (add nuclease digestion or precipitate the nucleic acid)
d. Inhibition of proteolytic enzymes in the sample (addition of protease inhibitors, rapid first step isolation at low temperature or expression of recombinant proteins in protease-deficient hosts)
(3) Storage conditions of the sample:
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
Longer storage (several days)
a, b, ibid.
c, adding appropriate bacteriostatic agents
Long-term storage
a, ibid.
b, frozen or preferably lyophilized (vacuum freeze-dried) preservation.
3. Establishment of evaluation methods for each purification step a. Determination of target protein content
Enzyme activity assay, biological activity assay, radioimmunoassay, enzyme-linked immunosorbent assay, immunoelectrophoresis, fluorescence.
b, total protein content determination
Ultraviolet absorption method, Lowry method, Bradford dye binding method (Coomassie brilliant blue G-250) and the like.
c, sample complexity detection
HPLC (ion exchange, gel filtration, reverse phase), SDS-PAGE, isoelectric focusing, capillary electrophoresis (CE).
4, the source of protein
(1) Natural protein: The target protein in the natural product is generally low in content and extremely complex in composition. In the separation process, multiple steps are required to remove various impurities, and the activity of the proteolytic enzyme should be reduced throughout the process.
(2) Recombinant protein: The recombinant protein has a high abundance of the target protein. There are three main expression orientations for recombinant proteins:
a, cytoplasm: in the case of species, the cell structure must be destroyed in order to obtain the protein of interest, accompanied by the release of a large number of nucleic acids and thousands of other host proteins; under the conditions of high expression, inclusion bodies are generally formed, inclusion bodies contain Overexpression of the protein of interest, and easy separation by high speed centrifugation, but the dissolution and renaturation of inclusion bodies is more complicated.
b. Periplasm: The expressed protein is present between the inner and outer membranes of the bacteria, so that the protein of interest can be less affected by proteases and reduce the contamination of host proteins.
c. Secretion to culture medium: This expression generally has a low protein concentration and is mainly contaminated by the medium.
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