General Considerations for Purification of GST-tagged Proteins

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

Yield of tagged protein is highly variable and is affected by the nature of the tagged protein, the host cell, and the expression and purification conditions used. Tagged protein yields can range from 1 mg/l up to 10 mg/l. Table 5.4 can be used to approximate culture volumes based on an average yield of 2.5 mg/l.

 

Table 5.4. Reagent volume requirements for different protein yields

Tagged protein yield 50 mg 10 mg 1 mg 50 µg
Culture volume 20 l 4 l 400 ml 20 ml
Volume of extract 1 l 200 ml 20 ml 1 ml
Glutathione Sepharose bed volume 10 ml 2 ml 200 µl 10 µl
1× PBS1 100 ml 20 ml 2 ml 100 µl
Glutathione elution buffer 10 ml 2 ml 200 µl 10 µl

1 This volume is per wash. Three washes are required per sample in the following procedures.

 

Use deionized (or double-distilled) water and chemicals for sample and buffer preparation. Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging the column; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.

One of the most important parameters affecting the binding of GST-tagged proteins to Glutathione Sepharose is the flow rate. Because the binding kinetics between glutathione and GST are relatively slow, it is important to keep the flow rate low during sample application to achieve maximum binding capacity. Washing and elution can be performed at a slightly higher flow rate to save time. For batch purification, incubation time should be considered.

The binding properties of the target protein can be improved by adjusting the sample to the composition of the binding buffer. Dilute in binding buffer or perform a buffer exchange using a desalting column (see Chapter 11, Desalting/buffer exchange and concentration).

Volumes and times used for elution may vary among tagged proteins. Further elution with higher concentrations of glutathione (20 to 50 mM) may improve yield. At concentrations above 15 mM glutathione, the buffer concentration should also be increased to maintain the pH within the range 6.5 to 8. Flowthrough, wash, and eluted material from the column should be monitored for GST-tagged proteins using SDS-PAGE in combination with Western blot if necessary.

Following the elution steps, a significant amount of tagged protein may remain bound to the medium. Volumes and times used for elution may vary among tagged proteins. Additional elutions may be required. Eluates should be monitored for GST-tagged protein by SDS-PAGE or by 1-chloro-2,4 dinitrobenzene (CDNB) assay for GST detection (see later in this chapter).

If monomers are desired, the GST tag should be cleaved off (see Removal of GST tag by enzymatic cleavage, Fig 5.20), because the GST-tagged protein can undergo dimerization.

Batch preparation procedures are frequently mentioned in the literature. However, the availability of prepacked columns and easily packed Glutathione Sepharose provides faster, more convenient alternatives. Batch preparations are occasionally used if it appears that the GST tag is not fully accessible or when the concentration of protein in the bacterial lysate is very low (both could appear to give a low yield from the affinity purification step). A more convenient alternative to improve yield is to decrease the flow  rate or pass the sample through the column several times (recirculation).

Purification steps should be monitored using one or more of the detection methods described later in this chapter. The GST Detection Module contains components that can be used for either enzymatic or immunochemical determination of concentrations of GST-tagged proteins in extracts as well as sample obtained during purification.

The yield of protein in purified samples can also be determined by standard chromogenic methods (e.g., Lowry, BCA, Bradford, etc.). If a Lowry or BCA type method is to be used, the glutathione in the purified material must be removed using, for example, a desalting column (see Chapter 11) or dialysis against 2000 volumes of PBS to reduce interference with the assay. The Bradford method can be performed in the presence of glutathione.

Reuse of purification columns and AC media depends upon the nature of the sample and should only be performed with identical samples to prevent cross-contamination.

Materials

     
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