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Protein Purification - Application Examples for Strep-Tactin®

The new Strep-Tactin®XT is the recommended purification resin of choice for Strep-tag®/Twin-Strep-tag® proteins.

Nevertheless in some cases purification of Strep-tag®/Twin-Strep-tag® proteins via Strep-Tactin® might be an alternative:

  • When elution of Strep-tag®/Twin-Strep-tag® proteins from Strep-Tactin®XT does not work
  • The target protein is a multimer
Strep-Tactin® Sepharose®

Unparalleled purity after one single purification step

After purification with the Strep-tag® system, there is no need for an additional purification step like size exclusion or ion exchange chromatography.

 

1: Sample of crude lysate after cytosolic expression with pASK-IBA3
2: Sample of flow through during chromatography
3: Sample after the addition of 2.5 mM desthiobiotin
M: Molecular size standard (kDa)

A 36 kDa enzyme and a mutant, both with C-terminal Strep-tag®II, were expressed in the cytoplasm of E. coli. The crude lysate was chromatographically separated on Strep-Tactin® Sepharose® (5 mg/ml) under gravity flow and physiological conditions (100 mM Tris-Cl, pH 8.0). The purification is documented on a Coomassie stained SDS gel where samples from the crude lysate (lane 1), from the flow through (lane 2), and from the elution with 2.5 mM desthiobiotin (lane 3) had been applied. The wild type enzyme is shown to be over 99 % pure (lane 3 contains 50 µg protein vs 0.5 µg protein per band in the molecular size standard). The mutant - although expressed at low level only - could be obtained at high purity under the same conditions.

Strep-Tactin® Superflow® (high cap)

Comparison of Strep-Tactin® Superflow® vs. Superflow® high capacity

Purification of Strep-tag®II proteins via Strep-Tactin® Superflow®

Strep-Tactin® Superflow® for intact virus like particle (VLP) 4.8 MDa purification

Purification of intact virus like particle using Strep-Tactin Supeflow
Figure 1, left to right: M; Rainbow Marker RPN 755
GE Healthcare Biosciences Corp.,
1 and 2; different elution fractions of VLPs
fused to Strep-tag II after one-step Strep-Tactin
affinity purification from a crude lysate of E. coli.

1 liter culture was induced at an OD600 of 0.6 using anhydrotetracycline (AHT) and protein expression was performed at 37°C for 3 hours at 200rpm. Cells were then pelleted, resuspended in 20 ml Buffer W (100 mM Tris-Cl, 150 mM NaCl, 1mM EDTA) and sonicated. The insoluble material was pelleted and the crude lysate was loaded on a Strep-Tactin® Superflow® column at a flow rate of 1ml/minute. After washing off the host proteins, the VLPs fused to Strep-tag® II were eluted using 2.5mM desthiobiotin in Buffer W. pASK-IBA7 was used for cloning.

For the purification of intact VLPs, the careful choice of both an ideal affinity tag system and the resin support is important:

  • The affinity chromatography process should be gentle in order to keep the VLPs intact, as with Strep-tag®.
  • The resin support packing within the column must allow penetration of the large VLPs. 

While MacroPrep® carriers did not meet criterion (2), since the viral particles accumulated on top of the column, Sepharose® and Superflow® enabled purification. Superflow® can be used in FPLC systems, whereas Sepharose® is only suitable for gravity flow purification.

These results were kindly provided by L. Stöckl and B. Brandenburg, Robert-Koch-Institute, Berlin.
Brandenburg B., Stockl L., Gutzeit C., Roos M., Lupberger J., Schwartlander R., Gelderblom H., Sauer I. M., Hofschneider P. H. and Hildt E. (2005)
A Novel System for Efficient Gene Transfer Into Primary Human Hepatocytes Via Cell-Permeable Hepatitis B Virus–like Particle, HEPATOLOGY (42),1300-1309
Strep-Tactin® MacroPrep®

For some recombinant proteins, purification characteristics on Strep-Tactin® MarcoPrep® differ from those on Strep-Tactin® Sepharose®

The Coomassie stained SDS gel shows the purification of a recombinant protein which exhibits exceptionally high non-specific protein binding. In this case, we have been able to remove the contaminants nearly quantitatively using the Strep-Tactin® MacroPrep® resin while the contaminants co-eluted with the recombinant protein after the same purification protocol using the Strep-Tactin® Sepharose® resin. (Please note: normally behaving recombinant proteins can be purified in a single step to homogeneity using Strep-Tactin® Sepharose®). This example shows that it may be reasonable to change the resin if non-specific protein binding occurs.

MacroPrep® is particularly advantageous, e.g. for plant proteins, where purification with MacroPrep® leads to better protein yields

Lane 1, molecular weight standard (kDa); Lane 2, Strep-Tactin® MacroPrep purified protein sample; Lane 3, Strep-Tactin® Sepharose® purified protein sample; Lane 4, soluble raw lysate as applied on each column

Protein purification using Strep-Tactin spin columns
lanes 1 and 8: marker;
lanes 2, 4 and 6: bacterial crude lysate;
lanes 3, 5 and 7: purified Strep-tag proteins

Strep-Tactin® Spin Column Purification of three different Strep-tag® proteins