Part:BBa_K627006
Fusion part of mdnA gene (from mdn-cluster) with myc-tag and gene III
This BioBrick is important for testing the suitability of a phage display system as an appropriate screening method for recombinant mdnA libraries.
The mdnA gene encodes the precursor peptide from the mdn-cluster, which is an essential building block for production of the protease inhibitor microviridin from Microcystis aeruginosa (Ziemert et al., 2008 and 2010). This peptide is known to be able to bind and inhibit proteases. Thus, variants of mdnA have a high potential for therapy as they can block disease-relevant proteases.
The gene III protein is a coat protein from the filamentous bacteriophage M13 (Smith, 1985). It appears only three to five times on the tip of the phage and is responsible for infection of bacterial cells. In phage display, genes from a DNA library are usually fused to the gene III and are displayed as fusion protein on the surface of the phages. Because it has been shown that the proteins of interest, which are fused to the C-terminus of gene-III can be functionally displayed (Krebber et al. 1997, Fuh and Sidhu, 2000, Rakonjac et al., 1999) only the sequence for this part has to be used.
The inserted sequence of the myc-tag enables easy detection and/or purification of the proteins of interest. In our case, the tag was used to verify display of the microviridin on the phage particle.
After transformation of E. coli with a vector containing the mdnA-myc-geneIII-fusion gene (in the context of the mdnA gene cluster) and co-infection with helper phages, E. coli cells were able to produce phage particles, which carry the microviridin peptide on their surface as confirmed by ELISA. These experiments proved the suitability of phage display as a screening method for variants of microviridin.
Detection of phages carrying mdnA on their surface by ELISA
The successful expression of the mdnA-myc-gene III-fusion protein on the surface of the phage was detected by ELISA. Therefore E. coli cells strains XL1-Blue and ER2738 were first transformed with the phagemid pPDV089 before they were infected with helper phages. E. coli cells containing both plasmids were selected. An ELISA test was performed to determine whether these cells are able to produce phage particles carrying the mdnA peptide on their surface. To perform this test anti-c-myc-antibodies were immobilized on ELISA plates and incubated with purified phages. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII protein of the phages. The HRP substrate o-phenyldiamine (OPD) was added and in case of binding a colour reaction was expected. The colour shift from achromatic to yellow in wells incubated with phages produced in XL1-blue cells showed the successful expression of mdnA-c-myc-gene III-fusion protein on the phages.In wells incubated with infected ER2738 cells no colour change was obserevd. That might be due to the fact that a mistake during phage purification was noticed.
For more precise results the absorption from 450 – 600 nm was measured. The data were presented in a bar plot. As a negative control helper phages were added instead of produced phages. Furthermore two wells were prepared were the secondary antibody was not added.
The graphic shows clearly the much higher absorption measured in wells, which were incubated with phage particles of interest produced in XL1-Blue cells. As has already pointed out this shows the succeeded expression of mdnA-c-myc-gene III-fusion protein on the surface of the phages.
Testing mdnA phage display and optimizing selection
To test the suitability of this screening method, phages representing unmodified mdnA on their surface and phages not representing mdnA (helper phages) in a ratio of one to one were incubated with immobilized trypsin which is known as a target of mdnA. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, E. coli cells XL1-Blue were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying mdnA are able to grow on agar with ampicillin whereas cells infected with helper phages are able to grow on agar with kanamycin. To control the success of the panning round additionally E. coli cells were infected with phage mix before panning and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells were infected with helper phages than with phages carrying mdnA despite of the engaged 1:1 ratio. This was surprising and indicated that mdnA on the surface of the phages may inhibit their infectivity. After controlling the plates an infection ratio of phages carrying mdnA to helper phages of 1:400 was calculated. This fact should be analyzed in further experiments.
The results of the first phage display are plotted in the figure below. After one panning round an enrichment of phages carrying mdnA was expected. This is attributable to the fact that phage particles carrying mdnA c-myc geneIII fusion protein on their surface are expected to bind specifically to the immobilized trypsin. Unfortunately this was not observed in this experiment. The ratio of cells growing on kanamycin agar before (4000) to cells growing on kanamycin agar (cells containing helper phages) after panning (750) was determined as 5:1. The ratio of cells growing on ampicillin agar before (12) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was nearly equal. Thus no enrichment of mdnA carrying phages occurred in the first experiment. So it was decided to repeat this experiment under improved conditions. Therefor the number of washing steps during the described experimental procedure was increased. Here the ratio of cells growing on kanamycin agar before (3000) to cells growing on kanamycin agar (cells containing helper phages) after panning (29) was determined as 103:1. The ratio of cells growing on ampicillin agar before (26) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was determined as 13:1. Thus an enrichment factor of eight was reached for the phages displaying mdnA on their surface.
These results indicate that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin. Therefore it can be deduced that mdnA is presented in a functional 3D structure. These findings suggest that phage display in general is an appropriate method for screening a recombinant mdnA library. Further experiments are required to optimize this system.
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