Intact VLP purification using Strep-Tactin Superflow: 4.8 MDa VLPs purified!
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 200 rpm. Then, cells were pelleted, resuspended in
20 ml Buffer W (100 mM Tris-Cl, 150 mM NaCl, 1 mM 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 1 ml/minute. After washing off the host proteins, the VLPs fused to Strep-tag II were eluted using 2.5 mM desthiobiotin in Buffer W. pASK-IBA7 was used for cloning.
Figure 2: Electron microscopy preparations of intact VLPs fused to Strep-tag II after purification on Strep-Tactin Superflow, uranyl acetate stain, 40 000x
* Strep-Tactin® Superflow® high capacity cartridges H-PR can directly be applied to
10-32 connections of HPLC and GE Healthcare ÄKTA™ Adapters for other workstations, syringes, peristaltic pumps as well as to connect several cartridges in series to enlarge capacity are listed in the order table above. |
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Specifications of Strep-Tactin Superflow resin
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Strep-Tactin® Superflow® |
Strep-Tactin® Superflow® high capacity |
| Binding capacity |
1 ml sedimented resin (corresponding to 2 ml of a 50 % suspension) can be used for the one-step purification of 50 to 100 nmol (up to 3 mg) recombinant protein |
1 ml sedimented resin (corresponding to 2 ml of a 50 % suspension) can be used for the one-step purification of 150 to 500 nmol or 3 -15 mg recombinant protein |
| Support |
Superflow 6 (6 % agarose, crosslinked) |
Superflow 6 (6 % agarose, crosslinked) |
| Bead size |
60-160 µm, spherical |
60-160 µm, spherical |
| Linear flow rate |
100-300 cm/h is recommended for Strep-tag purification |
100-300 cm/h is recommended for Strep-tag purification |
| Form (bulk) |
50 % suspension in 100 mM Tris/HCl pH 8.0,
150 mM NaCl, 1 mM EDTA |
50 % suspension in 100 mM Tris/HCl pH 8.0,
150 mM NaCl, 1 mM EDTA |
| Stability |
at least 6 months after shipment |
at least 6 months after shipment |
| Storage |
4°C, DO NOT FREEZE |
4°C, DO NOT FREEZE |
| Shipment |
RT |
RT |
Special Application
Strep-Tactin Superflow for intact virus like particle (VLP) purification
For the purification of intact VLPs the careful choice of both an ideal affinity tag system and the resin support is important:
1) The affinity chromatography process should be mild in order to keep the VLPs intact which is met by using Strep-tag.
2) The resin support packing within the column must allow penetration of the large VLPs.
While MacroPrep or POROS 20 carriers did not meet criterion (2), since in both cases the viral particles accumulated on top of the column, Sepharose and Superflow allowed the purification. Superflow can be used in FPLC systems whereas Sepharose is only suitable for gravity flow purification. These results, further illustrated in the figures on the left, were kindly provided by L. Stöckl and B. Brandenburg, Robert Koch Institute, Berlin.
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Protocols for download
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