Part:BBa_K245099

APH-p53

This part is designed to prepare a self-assembling polypeptide material which is composed of a homodimeric antiparallel coiled-coil segment (APH) and a p53 tetramerization domain linked by a dipeptide linker. This material forms a crosslinked lattice under the native conditions.

Part BBa_K245099 is intended to be cloned into the BioBrick vector under the strong promotor, such as T7. It has been experimentally in vector BBa_K245005

The gene for APH is of synthetic origin, optimized for expression in E. coli and based on aminoacid sequence of a designed homodimeric antiparallel coiled-coil from the reference (Gurnon et al., 2003). The gene for p53 tetramerization domain was also of synthetic origin, based on aminoacid sequence of human p53 tetramerization domain, optimized for expression in E. coli.

Assembly of part BBa_K245099

First, each of the coding sequences was cloned into the BioBrick vector (BBa_K245005). The vector already contains T7 promoter, RBS and ATG at 5' of multiple-cloning site, T7 terminator and STOP codon at 3' of multiple-cloning site. His-tag coding sequence is also incorporated between ATG and multiple-cloning site. Resulting basic parts were BBa_K245133 (APH inserted in functionalized vector BBa_K245005) and BBa_K245128 (p53 inserted in functionalized vector BBa_K245005).

The part with p53 was cut with NgoMIV and XbaI restriction enzymes and the part containing APH was cut with BspeEI and XbaI restriction enzymes. The APH-coding fragment was ligated into the linearized p53 part (BBa_K245128). Ligation resulted in a new composite part (BBa_K245099) where APH gene is located 5’ of p53. A joining of NgoMIV and BspEI restriction sites generates a protein-domain friendly scar tccggc (SG) after the ligation of basic parts BBa_K245133 and BBa_K245128. Resulting composite part BBa_K245099 has also His-tag-coding sequence due to design of our vector. Figure 1 shows the schematic representation of polypeptide product of this construct.

Figure 1: Scheme of the polypeptide product of construct APH-p53.

For protein synthesis E. coli BL21(DE3) pLysS transformed with part BBa_K245099 were cultivated and induced by the addition of IPTG. Protein was expressed in high yield (Figure 2) in form of inclusion bodies and hence easily isolated. His-tag linked to APH-p53 fusion enables protein detection using anti-His primary antibodies and its purification (Figure 2).

Figure 2: Western blot confirmation of expression of APH-p53 in E.coli.

The stability of the fusion protein APH-p53 was determined by far-UV spectra of this protein in solution (0.1 mg/ml) at different concentrations of GdnHCl (Figure 3).

Figure 3: Fraction of unfolded APH-p53 as a function of denaturant concentration.

This protein is a combination of tetramerization domain with homodimeric antiparallel coiled-coil-forming domain and hence is able to form a two-dimensional lattice. Membranes, generated in this way, have pores of defined dimensions that depend on the length of coiled-coil segment (APH in this case, but could be replaced with other coiled-coil segments, such as BBa_K245132). Schematic representation of a membrane assembly is shown on Figure 4. The formed membrane was investigated with scanning electron microscope and the microscopic image is shown on Figure 5.

Figure 4: Proposed assembly of a polypeptide consisting of a tetramerization domain and antiparallel homodimeric coiled-coil forming segment which results in formation of a polypeptide lattice with pores.

Figure 5: SEM image of APH-p53 (BBa_K245099) after dialysis of the inclusion bodies in MQ, before casting of the membrane.

We also demonstrated the functionality of these membranes by successful filtration of blue-dextran and bacteriophages. Results are demonstrated on Figures 6 and 7.

Figure 6: Filtration of blue-dextran. Blue-dextran (initial concentration 0.5 mg/ml) was retained by the APH-p53 membrane, which was determined by the absorbance of the filtrate at λ = 625 nm.

Figure 7: Clearance of bacteriophages by APH-p53 membrane. Comparison of viral titer before and after filtration through APH-p53 membrane.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]