Purify Full-Length Proteins Using Dual-Affinity Tags

Affinity tags, fused to the N-terminus or C-terminus of recombinant proteins, can be used for rapid, one-step affinity purification of proteins. Some recombinant proteins expressed in non-native hosts are produced as fragments due to incomplete translation or protease degradation. Fragments that retain the affinity tag can copurify with full-length protein. Dual affinity tags, fused to both the N-terminus and C-terminus of recombinant proteins, can be used for two-step purification of full-length proteins.1,2

This report demonstrates the advantages of dual affinity purification for two different proteins, Renilla luciferase (RLuc) and lipocortin I (LipI), which were cloned into the new Strep•Tag® II vectors, pET-51b(+) and pET-52b(+). Both vectors encode an N-terminal, Strep•Tag® II peptide sequence, for purification on Strep•Tactin® resin and a C-terminal His•Tag® peptide sequence for purification by immobilized-metal affinity chromatography (IMAC). Rosetta™ 2(DE3) Competent Cells, transformed with the recombinant vectors, were grown and autoinduced in Overnight Express™ Instant TB Medium. Cells were lysed using BugBuster® Master Mix.

His•Tag® fusion protein purification

Performing the initial purification by IMAC removes biotin and biotinylated proteins and concentrates the recombinant protein, which improves Strep•Tag® fusion protein binding in the second purification.1 Typically IMAC purification is optimal when the binding capacity of the resin is close to the concentration of the fusion protein. Because this will be the initial purification, the amount of resin need not be calculated and excess can be used to improve the yield. In this test, 10 mL soluble fraction was combined with 2 mL Ni-NTA His•Bind® Resin (4 mL 50% slurry) by batch method. Figure 1 shows the initial purification results for RLuc and LipI. Because purification was not optimized in this step, the resulting eluant contains nonspecifically bound proteins, as well as target protein fragments.

 

His•Tag® purification of RLuc and Lipl with C-terminal tags

Lane Sample
M Perfect Protein™ Markers
1 RLuc expressed in pET-51b(+)
2 RLuc expressed in pET-52b(+)
3 Lipl expressed in pET-51b(+)
4 Lipl expressed in pET-52b(+)

Figure 1. Purification of C-terminal His•Tag® fusion proteins. RLuc and Lipl were expressed in both pET-51b(+) and pET-52b(+) vectors and lysed as indicated in the text. IMAC purification was performed by combining 2 mL Ni-NTA His•Bind® resin with 10 mL soluble fraction for each sample and mixing at 4˚C for 15 min. Resin was washed with 20 mL 1X Ni-NTA Wash Buffer, and protein was eluted with 6 mL 1X Ni-NTA Elute Buffer. For each elution, protein concentrations were determined by BCA assay and 3 µg was assayed by SDS-PAGE and stained with RAPIDStain™ Reagent.

Strep•Tag® II fusion protein purification

Using Strep•Tactin® resin as the second purification step removes imidazole, which can cause protein aggregation, and allows the fusion protein to be eluted in a mild buffer, eliminating the need for a buffer exchange.1 Because the Strep•Tag®/Strep•Tactin® system is optimized for column purification, a column with 2 mL Strep•Tactin® resin was prepared and used to purify the fusion proteins eluted from the Ni-NTA His•Bind® resin. Figure 2 shows the dual-affinity purification results for RLuc and LipI. Nonspecific and degraded proteins are significantly reduced or removed after this step.

 

Strep•Tag® II purification of IMAC eluted RLuc and Lipl

Lane Sample
M Perfect Protein™ Markers
1 RLuc expressed in pET-51b(+)
2 RLuc expressed in pET-52b(+)
3 Lipl expressed in pET-51b(+)
4 Lipl expressed in pET-52b(+)

Figure 2. Purification of IMAC-eluted Strep•Tag® II fusion proteins. Columns were packed with 2 mL Strep•Tactin® resin equilibrated with 6 mL 1X Strep•Tactin® Wash Buffer. Ni-NTA His•Bind® elutions (6 mL) were loaded onto the columns. Columns were washed with 10 mL 1X Strep•Tactin® Wash Buffer. RLuc and Lipl fusion proteins were eluted with 6 mL 1X Strep•Tactin® Elution Buffer. For each sample, protein concentrations were determined by BCA assay and 3 µg was assayed by SDS-PAGE and stained with RAPIDStain™ Reagent.
 

Conclusion

Dual-affinity purification provides a quick and effective method to purify full-length fusion proteins. Using His•Tag® fusion protein purification followed by Strep•Tag® II fusion protein purification takes advantage of the benefits of both techniques and results in highly purified proteins.

 

Materials

Product No. Description
71553 pET-51b(+) DNA
71554 pET-52b(+) DNA
70666 Ni-NTA His•Bind® Resin
71592 Strep•Tactin® Superflow™ Agarose
69079 Perfect Protein™ Markers, 10-225 kDa
71400 Rosetta™ 2(DE3) Singles™ Competent Cells
71491 Overnight Express™ Instant TB Medium
71456 BugBuster® Master Mix
553215 RAPIDstain™ Reagent

 

References

  1. Cass, B., Pham, P.L., Kamen, A., and Durocher, Y. (2005) Protein Expr. Purif. 40, 77-85.
  2. Fiedler, M., Horn, C., Bandtlow, C., Schwab, M.E., and Skerra, A. (2002) Protein Eng. 15, 931-941.