Separation and purification of proteins by adsorption

Among the purification methods of proteins, selective adsorption is also one of the methods that have been widely used until now. Early work commonly used kaolin (a kind of soil in China, its molecular formula is roughly AL2O3 · 2SiO2 · 2H2O), alumina (AL2O3) and activated carbon and other adsorbents, because these adsorbents have weak adsorption, or easy to cause protein denaturation Widely, it has been replaced by gelled adsorbents, such as aluminum hydroxide gel and calcium phosphate gel, especially the latter.

There are two different ways of applying adsorbents. When the protein is more easily adsorbed, it is possible to select the appropriate conditions to mainly adsorb the protein and separate the impurities; when the protein is more difficult to adsorb, it is preferred to separate the impurities from the protein by facilitating the adsorption of impurities. Sometimes two methods can be used sequentially to achieve higher purification purposes. Adsorption conditions are usually carried out in slightly acidic conditions (pH 5 ~ 6) and dilute salt solution. Excessive salt concentration will interfere with the adsorption of proteins and enzymes, which means that a larger amount of adsorbent is needed to achieve certain results. The elution is generally carried out under weak base conditions or by appropriately increasing the ionic strength of the eluent to completely elute the adsorbed protein or enzyme.

The adsorption operation can be carried out by static adsorption or column chromatography, that is, the gel is packed into a column for adsorption operation. After the gel is packed, if the solution has poor passing ability, it can be mixed with a filter aid (such as diatomaceous earth) and a gel. When the ratio of the two is adjusted, a series of columns with different passing ability and adsorption capacity are obtained. Recently, hydroxyapatite [Ca10(PO4)6.(OH)2] has been isolated from proteins and enzymes, which can adsorb neutral and acidic proteins at high salt concentrations and exclude basic proteins. Adsorption is often carried out in phosphate buffer pH 6.8. When the elution is complete, the gel can be used repeatedly 3 to 4 times, and the gel with more impurities is washed with 0.1 mol/L sodium hydroxide or 1 mol/L hydrochloric acid.

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