Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp

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Soluble ZFPs


Figure 7: Three dimensional representation of chimeric ZFP. MBP is shown in green and ZFP (Zif268) is shown in cyan, bound to the target DNA (orange and blue).
A need for properly folded and soluble zinc finger proteins was central for the success of our project. Our colleagues at iGEM2010 competition used zinc fingers for positioning proteins on the linear DNA within bacterial cells and did not face the problem of solubility and propensity of ZFPs to aggregate in vitro. Limited with the time frame of the competition we decided to search for the universal solution other than trying to optimize the solution conditions for each ZFP separately.

It has been known from the literature that fusion of proteins that have a tendency to aggregate with highly soluble protein often results in rescuing the "misbehaved proteins". Therefore we fused zinc finger proteins with highly soluble protein domains, namely GST (glutathione-S-transferase) and MBP (maltose binding protein). Preparation of cloning constructs (which was however not straightforward and without problems) is described in more detail in Plasmid construction section in Methods.

During BIOMOD2011 project we successfully achieved bacterial expression of 14 chimeric zinc finger proteins and isolated 11 of them, all of which had added solubility domains or tags. Use of solubility tags allowed us to isolate chimeric zinc finger proteins under the non-denaturing conditions from bacterial lysates, which preserved the biological function of isolated proteins (described in the section on Functionalized ZFPs).

We produced six zinc finger protein chimeras tagged with GST. Three of them, GST-Zif268, GST-sZif268 and GST-PBSII, contained three-finger domains recognizing 9 base pairs of their DNA targets. On Figure 8 we present the analysis of soluble and insoluble fractions of bacterial lysates containing chimeric ZFPs (detailed methods are described in Protein expression of the Methods section).

Two of the proteins, GST-Zif268 and GST-sZif268, were also successfully purified by chelating chromatography as shown in Figure 9 of SDS-PAGE and Western blot analysis.
Figure 8: Production of GST-fused three-finger domain-containing ZFPs. Each image comprises samples from the soluble fraction of bacterial lysate (SN or supernatant) and insoluble fraction (IBs or inclusion bodies). Left to right: GST-Zif268 (Mr of 38 kDa), GST-sZif (Mr of 38 kDa), GST-PBSII (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies. Figure 9: Purification of GST-fused three-finger domain containing ZFPs. Each image shows a sample of isolated proteins GST-Zif268 (Mr of 38 kDa) and GST-sZif (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: western blot analysis using anti-His4 antibodies.

Additionally, we isolated three chimeric proteins tagged with GST: GST-2C7, GST-AZPA4 and GST-6F6. Each of these ZFPs contains six-fingers, thus recognizing 18 base pair target DNA. Production of six-finger chimeras was confirmed by SDS-PAGE and Western blot analysis, which can be seen in Figure 10. All of them were successfully purified as shown in Figure 11.
Figure 10: Bacterial production of GST-fused six-finger domains containing ZFPs. Each image comprises samples from bacterial lysate soluble fraction (SN or supernatant) and insoluble fraction (IBs or inclusion bodies). Left to right: GST-AZPA4 (Mr of approximately 47 kDa), GST-2C7 (Mr of approximately 49 kDa), GST-6F6 (Mr of approximately 48 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies. Figure 11: Purification of GST-fused six-finger domains containing ZFPs. Each image shows a sample of isolated proteins GST-AZPA4 (Mr of approximately 47 kDa), GST-2C7 (Mr of approximately 49 kDa) and GST-6F6 (Mr of approximately 48 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies.

6F6 and 2C7 were also fused with MBP. Production and isolation of MBP-6F6 and MBP-2C7 was analyzed by SDS-PAGE and Western blot, which can be seen in Figure 12.
Figure 12: Production and isolation of MBP-fused ZFPs containing six-finger domains. Left to right: Coomassie stained SDS-PAGE gels of soluble and insoluble fractions of lysates of bacteria producing MBP-6F6 (Mr of approximately 64 kDa) and MBP-2C7 (Mr of approximately 65 kDa); Western blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions using anti-His4 antibodies; Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mr of approximately 64 kDa); Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mr of approximately 65 kDa).

After initial failed attempts to produce soluble zinc finger protein chimeras with mCitrine and Renilla luciferase proteins, we decided to add MBP to their N-termini. We confirmed that the addition of MBP rendered such chimeric proteins more soluble and readily expressed in the functional and soluble form.

We used the same reasoning for the production of twin zinc finger protein chimeras, that is 2C7-MBP-6F6 and AZPA4-MBP-6F6 chimeric proteins which were designed for the vertical DNA origami stacking using protein tethers. In this case, solubility was also increased by the addition of MBP domain, nevertheless it was somewhat lower than that for other proteins described in this section, which might be due to the fact that the aggregation propensity of two zinc finger domains could not be sufficiently compensated by the addition of a single solubility domain.
 

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