Difference between revisions of "Team:Wageningen UR/Results/Phage Display"
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| − | <p>Phage display is a powerful laboratory technique used to characterize protein interactions and functions. In phage display, large libraries can be used to select for proteins with desired properties. Using M13 phage display, we can select for the best affinity molecule against chikungunya virus (CHIKV) and Human African Trypanosoma | + | <p>Phage display is a powerful laboratory technique used to characterize protein interactions and functions. In phage display, large libraries can be used to select for proteins with desired properties. Using M13 phage display, we can select for the best affinity molecule against chikungunya virus (CHIKV) and Human African Trypanosoma (HAT) antigens. |
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| − | In M13 phage display, the DNA sequence encoding | + | In M13 phage display, the DNA sequence encoding the protein of interest (in our case the affinity body library) is fused to a gene which codes for the M13 phage G3P minor coat protein. After correct assembly, the phage will display the affinity body on its surface, linking the genotype to the phenotype. To fuse the affinity body to the phage coat protein, two distinct strategies can be applied. The first method is to clone the affinity body sequence directly into the phage genome, in the coding sequence of the coat protein (phage vector). The other option is to use a small vector (phagemid vector) coding for part of the phage G3P minor coat protein. The protein of interest can be cloned into the phagemid vector and will be expressed as a fusion with G3P (Figure 2). The advantage of using phagemids is the ease of cloning into the smaller vector, and efficiency is important during the cloning of libraries. Furthermore, using a phagemid results in a monovalent display of the protein of interest compared to a polyvalent display with the phage vector (Paschke et al. 2009). This is an important difference since a polyvalent display could lead to the selection of weak-binding variants while this is circumvented with a monovalent display. However, the phagemid vector approach must be used in combination with a helper phage. In our case we used the VCSM13 helper phage (Figure 2) which is a M13-based phage and has a defective origin of replication. The phagemid vector has a working origin of replication and leads to the preference for packing the phagemid vector in the phage particles. With all this in mind we chose for the phagemid vector approach. |
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| − | The ligation of the library into the pComb3XSS vector has been described <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Affinity_Bodies">here</a>. After ligation, the mixture was electroporated into XL1-Blue E.coli cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture 250 mL of fresh medium was added and grown in 1L flasks until an | + | The ligation of the library into the pComb3XSS vector has been described <a href="https://2017.igem.org/Team:Wageningen_UR/Results/Affinity_Bodies">here</a>. After ligation, the mixture was electroporated into XL1-Blue <i>E. coli</i> cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture 250 mL of fresh medium was added and grown in 1L flasks until an OD<sub>600</sub> of ~0.6 was reached. VCSM13 helper phages were added at a MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity. The resulting recombinant phage library has an infective titer of 5.7*10<sup>15</sup> CFU/mL (colony forming units per mL). This library can then be used to screen for the appropriate affibody against any antigen. |
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| − | Antigens were produced for CHIKV, Zika virus (ZIKV) and | + | Antigens were produced for CHIKV, Zika virus (ZIKV) and mayaro virus (MAYV) in the form of VLP’s. Strep-tagged surface proteins were produced for CHIKV and HAT. In order to purify the VLP’s and attach them to beads, antigens for the specific virus particles can be linked to protein-A resin beads, washed, blocked and used for phage display. However, due to the cost of the antigens and required resin beads, we chose to continue with only the strep-tagged surface proteins. |
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| − | <br> The beads containing the CHIKV and HAT antigens were washed and subsequently blocked using PBS-Tween with Gelatin. Milk or BSA was not used for blocking due to the possible presence of immunoglobulins which is the natural target of our | + | <br> The beads containing the CHIKV and HAT antigens were washed and subsequently blocked using PBS-Tween with Gelatin. Milk or BSA was not used for blocking due to the possible presence of immunoglobulins which is the natural target of our wildtype affinity body. Next, the phage library was added and after incubation at room temperature, the unbound phages were washed away using PBS-Tween. The phages still bound to the beads were eluted, titrated and amplified. After amplification the phages were titrated and the experiment/binding affinity assay was repeated. Stringency was increased with each round of panning, by increasing Tween concentration during the washing step. Tween concentrations in round 1,2, and were 0.1, 0.2% and 0.4% respectively. The exact protocols used can be found <a href="https://2017.igem.org/Team:Wageningen_UR/Notebook/Protocols">here</a>. |
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Revision as of 10:49, 1 November 2017
Phage Display
Phage display is a powerful laboratory technique used to characterize protein interactions and functions. In phage display, large libraries can be used to select for proteins with desired properties. Using M13 phage display, we can select for the best affinity molecule against chikungunya virus (CHIKV) and Human African Trypanosoma (HAT) antigens.
Generation of recombinant M13 phages
In M13 phage display, the DNA sequence encoding the protein of interest (in our case the affinity body library) is fused to a gene which codes for the M13 phage G3P minor coat protein. After correct assembly, the phage will display the affinity body on its surface, linking the genotype to the phenotype. To fuse the affinity body to the phage coat protein, two distinct strategies can be applied. The first method is to clone the affinity body sequence directly into the phage genome, in the coding sequence of the coat protein (phage vector). The other option is to use a small vector (phagemid vector) coding for part of the phage G3P minor coat protein. The protein of interest can be cloned into the phagemid vector and will be expressed as a fusion with G3P (Figure 2). The advantage of using phagemids is the ease of cloning into the smaller vector, and efficiency is important during the cloning of libraries. Furthermore, using a phagemid results in a monovalent display of the protein of interest compared to a polyvalent display with the phage vector (Paschke et al. 2009). This is an important difference since a polyvalent display could lead to the selection of weak-binding variants while this is circumvented with a monovalent display. However, the phagemid vector approach must be used in combination with a helper phage. In our case we used the VCSM13 helper phage (Figure 2) which is a M13-based phage and has a defective origin of replication. The phagemid vector has a working origin of replication and leads to the preference for packing the phagemid vector in the phage particles. With all this in mind we chose for the phagemid vector approach.
The ligation of the library into the pComb3XSS vector has been described here. After ligation, the mixture was electroporated into XL1-Blue E. coli cells. The colonies on the transformation plates were scraped together and suspended in 50 mL of 2xYT medium supplemented with 1% (w/v) glucose and 100 ug/mL ampicillin. To this culture 250 mL of fresh medium was added and grown in 1L flasks until an OD600 of ~0.6 was reached. VCSM13 helper phages were added at a MOI of 20 and incubated at 37°C without shaking. After incubation, the medium was changed to a 2xYT medium supplemented with 1% (w/v) glucose, 100 ug/mL ampicillin, 50ug/mL kanamycin and 0.25mM IPTG and incubated overnight at 30°C. The next day the phages were precipitated and a titration was performed to assess infectivity. The resulting recombinant phage library has an infective titer of 5.7*1015 CFU/mL (colony forming units per mL). This library can then be used to screen for the appropriate affibody against any antigen.
Selection
Antigens for CHIKV and HAT were attached to Streptavidin-Sepharose resin beads for phage display (mentioned above). The beads were exposed to the phage library, washed and then eluted. The eluted phage was then amplified and used again for a second and third round of biopanning , increasing the stringency of the wash with each step. In the last step, the eluted phage is used to infect XL1-Blue cells. Colony PCR is then used to amplify the phagemids and sent for sequencing to confirm the sequence of the best binding affinity molecule (Figure 3).
Antigens were produced for CHIKV, Zika virus (ZIKV) and mayaro virus (MAYV) in the form of VLP’s. Strep-tagged surface proteins were produced for CHIKV and HAT. In order to purify the VLP’s and attach them to beads, antigens for the specific virus particles can be linked to protein-A resin beads, washed, blocked and used for phage display. However, due to the cost of the antigens and required resin beads, we chose to continue with only the strep-tagged surface proteins.
The beads containing the CHIKV and HAT antigens were washed and subsequently blocked using PBS-Tween with Gelatin. Milk or BSA was not used for blocking due to the possible presence of immunoglobulins which is the natural target of our wildtype affinity body. Next, the phage library was added and after incubation at room temperature, the unbound phages were washed away using PBS-Tween. The phages still bound to the beads were eluted, titrated and amplified. After amplification the phages were titrated and the experiment/binding affinity assay was repeated. Stringency was increased with each round of panning, by increasing Tween concentration during the washing step. Tween concentrations in round 1,2, and were 0.1, 0.2% and 0.4% respectively. The exact protocols used can be found here.
Results
References
- Paschke, Matthias. "Phage display systems and their applications." Applied microbiology and biotechnology 70.1 (2006): 2-11.
