Part:BBa_K4674013

The PBSII-Zif268 expression cassette

This expressing cassette is regulated by lac operator. The N-terminal of PBSII-Zif268 protein is fused with a 6xHis-tag and a thrombin cleavage site.

ZFP proteins: the zinc finger binding domain from PBSII and Zif268

Zinc figer protein (ZFP) are abundantly expressed and have diverse functions. ZFP can interact with DNA, RNA, and proteins. The classic zinc finger structure is maintained by zinc ion, which coordinates cysteine and histidine in different combinations. Importantly, the zinc finger shows DNA binding specificity. In our design, we apply the zinc finger domain form Zif268 (Bulyk et. al. 2001) and PBSII (Conrado, R. J., et al. 2012) for DNA tetrahedron docking.

Zif268 is a naturally occurring 3-finger ZF that has been extensively studied structurally and biochemically. It binds the 9 bp sequence 5′-GCG TGG GCG-3′. PBSII is designed 3-finger ZFs assembled from predefined modified ZF domains, and recognize the sequences 5′-GTG TGG AAA-3′

Experimental result

After confirming the cloning, we induced the PBSII-Zif268 fusion protein expression by treating 0.25 mM IPTG at 37°C. The PAGE analysis showed that the fusion protein was located in the inclusion body (P lane). We then modified the induction temperature to 16°C, however, the PAGE analysis result still showed the inclusion body location of PBSII-Zif268 fusion protein.

Fig. The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein induced at different temperatures and IPTG concentrations. Sup: supernatant; P: pellet

We then try to extract the PBSII-Zif268 fusion protein from the inclusion body by 8 M Urea solution. The PAGE analysis showed that the treatment of 8 M Urea could solubilize the PBSII-Zif268 fusion protein (the Sup 2 lane).

Fig. The coomassie blue staining of SDS-PAGE analysis of PBSII-Zif268 fusion protein extracted from the inclusion body. Sup 2: the urea-soluble supernatant; P2: the urea-insoluble pellet

Future work

The 8 M Urea extracted PBSII-Zif268 fusion protein needs to be purified by Ni2+ bead and renature into the functional structure for docking DNA tetrahedron. We have found the protocol for protein renature on the Ni2+ beads or after elution. The corresponding experiments will be performed to optimize the PBSII-Zif268 fusion protein collection.

Reference

Bulyk, M L et al. (2001) Exploring the DNA-binding specificities of zinc fingers with DNA microarrays Proc Natl Acad Sci U S A. 19;98(13):7158-63.

Conrado, R. J., et al.(2012). DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic acids research, 40(4), 1879–1889.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 424
  Illegal BamHI site found at 711
  Illegal XhoI site found at 706
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  
• 1000
  COMPATIBLE WITH RFC[1000]