General Considerations for Purification of GST-tagged Protein

The yield of GST-tagged proteins is highly variable, ranging from 1 mg/l to 10 mg/l. The yield depends on various parameters, such as nature of the tagged protein, the host cell, and the culture conditions used. Table 3.2 shows cell culture, medium, and buffer volumes for obtaining an average yield of 2.5 mg/l.

Table 3.2 Recommendations on reagent volumes and culture size

Tagged protein yield	50 mg	10 mg	1 mg	50 µ g
Culture volume	20 l	4 l	400 mL	20 mL
Volume of lysate	1 l	200 mL	20 mL	1 mL
Glutathione Sepharose bed volume	10 mL	2 mL	200 µL	10 µL
Volume of wash buffer	300 mL	60 mL	6 mL	300 µL
Glutathione elution buffer	10 mL	2 mL	200 µL	10 µL

One of the most important parameters affecting the binding of GST-tagged proteins to Glutathione Sepharose media 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 slightly higher flow rates. For batch purification, the incubation time should be considered.

Use deionized or double-distilled water and high-grade 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 clogging of the column, or perform an efficient lysis treatment, for example, by sonication and homogenization. DNase/RNase can be added to the sample to reduce the size of nucleic acid fragments.

The binding properties of the target protein can be improved by diluting the sample in binding buffer or performing a buffer exchange using a desalting column such as PD-10 Desalting Columns, HiTrap Desalting 5 mL, or HiPrep 26/10 Desalting.

Volumes and times used for elution may vary for different tagged proteins. Further elution with higher concentrations of glutathione (20 to 50 mM) may improve the 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 fractions from the column should be monitored for detection of the GST-tagged protein using SDS-PAGE, in combination with Western blotting, or CDNB assay if necessary.

After the elution steps, there might still be some remaining tagged protein bound to the medium. Additional elutions may be required.

If monomers are desired, the GST tag should be cleaved off. Gel filtration of the GST-tagged protein will probably give an unstable preparation of GST-tagged monomers that will immediately start to form dimers via GST-GST interactions.

Batch preparation procedures are frequently mentioned in the literature, but the availability of prepacked columns and easily packed Glutathione Sepharose provides faster and 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, giving low yields from the affinity purification step. A more convenient alternative to improve the 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. The GST Detection Module contains components that can be used for enzymatic or immunochemical determination of GST-tagged protein concentrations.

The yield of the purified tagged protein can also be estimated by measuring the absorbance at 280 nm or by standard chromogenic methods (e.g., Lowry, bicinchoninic acid [BCA], Bradford, etc.). The Bradford method can be performed in the presence of glutathione, but when a Lowry or BCA type method is used, the glutathione in the purified material must be removed using a desalting column or dialysis against 2000 volumes of PBS to reduce interference with the assay.

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