Self-assembling membranes Results.html

The genes encoding protein domains were fused according to our BioBrick standard. The proteins APH1-p53, APH-p53, p53-APH, BCR-p53 and p53-BCR were expressed in E. coli BL21(DE3) pLysS mostly in form of inclusion bodies (Figure 1, Figure 2, Figure 3).  

Figure 1: Production and purification of APH1-p53. SDS-PAGE analysis of the cell lysate supernatant (lane 1), insoluble fraction (lane 2), and purified polypeptide (lane 3). The band of calculated size of the polypeptide APH1-p53, 8.44 kDa is visible and was confirmed by mass spectrometry.

Figure 2: Production of BCR-p53 and p53-BCR. SDS-PAGE analysis of BCR-p53 (lane 1) and p53_BCR (lane 2) insoluble cell lysate show that proteins were expressed in form of inclusion bodies.

Figure 3: Western blot of APH-p53. APH-p53 is predominantly expressed in form of inclusion bodies as confirmed by Western blot using anti-His-tag antibodies (lane 2) in contrast to soluble fraction of cell lysate (lane 1). The third line represents protein standard.

We used circular dichroism spectroscopy to check whether proteins possess the expected alpha-helical secondary structure and to determine their stability.  Far-UV CD spectra of all tested proteins show two minima, at 208 nm and 222 nm, which is characteristic for alpha-helical secondary structure (Figure 4).

Figure 4: Far-UV CD spectrum of APH1-p53 confirmed predominant alpha-helical secondary structure. Similar spectra were obtained for other constructs.  

Figure 5: Fraction of unfolded APH-p53 as a function of denaturant concentration. Far-UV CD spectra of APH-p53 (0.1 mg/ml) were measured in solution with different concentrations of GdnHCl

Dialysis of solubilized APH-p53 inclusion bodies already produced a membrane-like material. The scanning electron microscopy (SEM) image of this membrane showed a porous material (Figure 6). Several fissures visible on image are probably due to non-optimal preparation of the sample with vacuum drying.

Figure 6: SEM image of APH-p53 membrane-like material

The following protocol was used to prepare the membrane by self-assembly of fusion polypeptide (Figure 7). Polypeptide was dissolved in 9M LiBr and then refolded by dilution into 20mM HEPES so the final concentration of protein in suspension was 0.2 mg/ml. 500 µl of protein suspension was slowly deposited on a filter with 0.2 µm pores with liquid being constantly removed by gentle vacuum (Figure 7). After deposition of the assembled protein to the support membrane, 10 % of glutaraldehyde was added to covalently crosslink and stabilize the protein assembly. After one hour, the membrane was rinsed with MQ water. The step of deposition and crosslinking of the proteins was repeated if required.

Figure 7: Flow chart of the protocol for membrane preparation.

Figure 8: The filtration system. Details of filtration system are depicted in the circle.

Blue dextran solution (0.5 mg/ml), and M13 bacteriophages were filtered through the prepared membrane to check its permeability. Concentrations of blue dextran and bacteriophage titer in the flow-through were determined. When we compared the concentrations and titer before and after the filtration we obtained encouraging results. The protein membrane retained most of the blue dextran (Figure 8) and bacteriophages (Figure 9).

Figure 9: Filtration of blue dextran. Blue dextran with initial concentration of 0.5 mg/ml did not flow through the APH-p53 membrane as was determined by measuring of the filtrate absorbance at 625 nm.

Figure 10: Clearance of bacteriophages by APH-p53 membrane. A) Comparison of viral titer before and after filtration through APH-p53 membrane. B) Scheme shows that ordinary filter can not retain the virus, while the addition of polypeptide membrane above the filter support does.