Preparative Purification using GSTPrep™ FF 16/10 column

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

GSTPrep™ FF 16/10 columns are based on the 20 ml HiPrep column design, ready to use for easy, one-step preparative purification of GST-tagged proteins, other glutathione S-transferases, and glutathione binding proteins. Prepacked with Glutathione Sepharose® 4 Fast Flow, the columns exhibit high binding capacity and excellent flow properties. For easy scale-up, columns can be connected in series.

GSTPrep FF 16/10 column.

Fig 5.13. GSTPrep™ FF 16/10 column.

 

The column is made of polypropylene, which is biocompatible and noninteractive with biomolecules. Separations can be easily achieved using a chromatography system such as ÄKTA. Refer to Table 2.1 in Chapter 2 (Manual and automated purification) for a selection guide to purification equipment and to Appendix 2 (Characteristics of Glutathione Sepharose® products) for a list of GSTPrep™ FF 16/10 column parameters.

Glutathione Sepharose® 4 Fast Flow is also available as prepacked 1 ml and 5 ml GSTrap FF columns, and as a bulk medium in bulk packs (25, 100, and 500 ml) for packing columns or batch purifications. Note that GSTPrep™ FF 16/10 columns cannot be opened or refilled.

 

Sample preparation

Refer to General considerations for purification of GST-tagged proteins for general considerations before beginning this procedure.

Adjust the sample to the composition and pH of the binding buffer by additions from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.

Pass the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before sample application. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.


Buffer preparation

Use high-purity water and chemicals, and pass all buffers through a 0.45 µm filter before use.

Binding buffer: PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.3
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

1 to 20 mM DTT may be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups on GST, which may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.


Purification

  1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of 1 to 5 ml/min (30 to 150 cm/h).
  2. Wash the column with 100 to 200 ml of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
  3. Elute the bound protein with 100 to 200 ml of elution buffer at a flow rate of 2 to 10 ml/min (60 to 300 cm/h).
  4. Equilibrate the column with 60 to 100 ml of binding buffer at a flow rate of 2 to 10 ml/min (60 to 300 cm/h). The column is now ready for a new purification.

Due to the relatively slow binding kinetics between GST and glutathione, it is important to keep the flow rate low during sample loading/elution. The binding capacity may be different for different proteins. The yield may therefore vary between proteins if sample load is close to the capacity of the column.

Optional: Collect the flowthrough and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to check for any loss of unbound target protein.

Reuse of any purification column depends on the nature of the sample and should only be performed with identical tagged proteins to prevent cross-contamination.

For cleaning, storage, and handling information, refer to Appendix 2 (Characteristics of Glutathione Sepharose® products).

 

Application example

Purification and scale-up of two GST-tagged proteins using 1 ml and 5 ml GSTrap FF  columns and GSTPrep™ FF 16/10 column

Glutathione Sepharose® 4 Fast Flow is easy to use for one-step purification of GST-tagged proteins. Figures 5.14A-C and 5.15A-C show scale-up studies on GSTrap™ FF 1 ml, GSTrap™ FF 5 ml, and GSTPrep™ FF 16/10. Two different GST tagged proteins were purified: GST-DemA and GST-Purα. The gene encoding for DemA was isolated from Streptococcus dysgalactiae. DemA is a fibrinogen-binding protein that shows both plasma protein binding properties and sequence similarities with the M and M-like proteins of other streptococcal species. Purα has been shown to be involved in transcriptional regulation.

E. coli expressing the GST-tagged proteins was resuspended (1 g/5 ml) in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) supplemented with 1 mM PMSF, 1 mM DTT, 100 mM MgCl2, 1 U/ml RNase A, and 13 U/ml DNase I. The cells were lysed by sonication with a Vibracell™ ultrasonic processor for 3 min, amplitude 50%. The cell extract was kept on ice during the sonication. Cell debris was removed by centrifugation at 48 000 × g, 4°C for 30 min. The supernatant was applied to the column after passage through a 0.45 µm filter.

The following purification procedures were performed using an ÄKTA chromatography system. The columns, GSTrap™ FF 1 ml, GSTrap™ FF 5 ml, and GSTPrep™ FF 16/10 were equilibrated with 5 column volumes of PBS, pH 7.4, and the prepared sample was applied to the columns.

The columns were washed with 10 column volumes of PBS (GST-DemA) and 20 column volumes

of (GST-Purα) and eluted using 7 column volumes of Tris-HCl, pH 8.0 including 10 mM reduced glutathione. The purity of eluted proteins was analyzed by SDS-PAGE (see Figs 5.14D and 5.15D).

The main parameter in this scale-up study was the residence time (i.e., the period of time the sample is in contact with the chromatography medium). The residence time was the same for the GSTrap™ FF 1 ml and 5 ml columns whereas it was twice as long for the GSTPrep™ FF 16/10 column (20 ml column volume) compared with GSTrap FF 5 ml columns due to the difference in column length vs column diameter. The amount of protein bound differed between GST-DemA and GST-Purα, due to protein-dependent binding characteristics. Some of the applied protein was found in the flowthrough as an effect of the slow binding kinetics of GST. The amount of eluted GST-tagged proteins increased proportionally with increased column volume and sample load.

 Purification and scale-up of GST-DemA

SDS-PAGE analysis of GST-DemA

Fig 5.14. Purification and scale-up of GST-DemA on (A) GSTrap FF 1 ml, (B) GSTrap FF 5 ml, and (C) GSTPrep™ FF 16/10. (D) SDS-PAGE analysis of GST-DemA on ExcelGel Homogeneous 12.5% using Multiphor™ II followed by Coomassie staining. Due to the relatively slow binding kinetics of GST and rather high load, some of the applied protein was found in the flowthrough.

 

 

Purification and scale-up of GST-Purα

SDS-PAGE analysis of GST-Purα

Fig 5.15. Purification and scale-up of GST-Purα on (A) GSTrap FF 1 ml, (B) GSTrap FF 5 ml, and (C) GSTPrep™ FF 16/10. (D) SDS-PAGE analysis of GST-Purα on ExcelGel Homogeneous 12.5% using Multiphor II followed by Coomassie staining. Due to the slow binding kinetics of GST, some of the applied protein was found in the flowthrough.

Materials

     
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