The Curious Cloning Conundrum

CASE STUDY 1- THE CURIOUS CLONING CONUNDRUM

Antibodies are immune system-related proteins, having a unique target on an invading organism (Cohen, 1975). This site has to be recognised and targeted, in order to activate our immune system to produce specific antibodies against it. The aim of the following case study is to design a polyclonal antibody that would recognise particular sites in all the E2F4 proteins. The identical amino acid sequences or homologous regions to which the antibody binds with a high affinity, common to all E2F proteins, could be determined by using Clustal Omega (Li, Cowley et al., 2015), a bioinformatics tool which is used for multiple alignment of large number of protein sequences (Fig.1).

1. Multiple sequence alignment:

[pic 1]

Fig.1- Clustal Omega results for multiple sequence alignment of E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6 proteins. blue, region of interest; (*), with identity; (:), high similarity; (-), gap.

2. Antigenicity plot: [pic 2]

The multiple alignment sequences from Clustal omega yielded homologous sequences and the most identical and similar sequences were used for antigenicity plot using Kolaskar & Tongaonkar bioinformatics tool(Ponomarenko & Bourne, 2007). The E2F4 sequence that showed highly similar amino acids is RHEKSLGLLTTKFVSLLQEAKDGVLDLKLAADTLAVRQKR RIYDITNVLEGIGLIEKKSKNSIQWKG. The following region KRRIYDITNVLEGIGLI, containing the predicted peptide ITNVLEGIGLI of 11 amino acids along was selected and chosen as it had a threshold value above 1.000 and has the hydrophobic amino acids valine and leucine, which can act as an effective antigenic site (Kolaskar & Tongaonkar, 1990). The extra amino acids KRRIYD are used, in order to increase the surface area. In addition to it, this sequence is found in all the 6 E2F proteins.

1. 3D representation of the selected antigenic region:

[pic 3]

VMD representation gives a 3D image of the E2F4 protein crystal structure and is used to visualise the cloning region . The selected region KRRIYDITNVLEGIGLI can act as an epitope as it is more exposed to the surface of the E2F4 protein and therefore, can function as an effective surface antigen recognition site for the polyclonal antibody to bind to it (Fig 3). As the amino acid stretch YDITNVL is present and is homologous in all the E2F4 proteins, it can provide a high binding efficiency as an E2F-TDP domain is present in that region in all the E2F proteins and binds DP to nucleic acids. Therefore, it can act an antigenic determinant.

Fig 3. 3D representation of E2F4 protein- (a) Selected antigenic region, yellow (b) Small stretch of homologous region to all E2F4 proteins, blue (c) All nucleic, Green (d) All protein, Orange.

1. Functional information:

[pic 4]

Fig 4. Functional domain of E2F4 showing the E2F-TDF domain in green

The functional interaction of the protein can be found using SMART program (Letunic, Doerks et al., 2015). Furthermore, PSORT was used to predict the localisation of E2F4 protein which was predicted to be 56.5% localised in the nuclear region (Brameier et al, 2007). The E2F transcription factor dimerises with TDP1 and TDP2 and stimulates the E2F dependent transcription . It then binds to DNA either as a homomer or heteromer and therefore increases the binding efficiency. The E2F/DP family have winged-helix DNA binding motifs which are involved in expression of genes regulating cell cycle and in stimulating E2F-dependent transcription.