Team:Wageningen UR/Results/Adhesion Library
Adhesion Library
One of the assets of the Xylencer phage are its adhesion proteins: proteins, fused to the capsid of the phage that bind to the chitin stylets of insects, resulting in the spread of the Xylencer phage in an infected area. The aim of this subproject is to identify potential candidates for this adhesion protein and to test their chitin binding capacities. After extensive literature research, the candidates were expressed in E. coli BL21DE3 and purified with the fused strep-tag. After isolation, chitin binding assays were performed to test the chitin binding. Xylella fastidiosa hypothetical protein PD1764, with its first 20 amino acids removed (PD1764sh), deemed to be a good candidate for this capsid fusion and application on the Xylencer phage.
Introduction
Chitin is a polymer that consists out of N-acetyl-1,4-beta-D-glucopyranosamine monomers. It is
found in the
cell walls of fungi and in the exoskeleton of arthropods, such as insects, spiders and
crustaceans,
where it improves the sturdiness [1].
A lot of plant pathogens (both bacteria and viruses)
use these
chitin residues/moieties to spread. The bacteria and viruses have chitin binding proteins on
their
membrane/capsid and attach themselves to the chitin residues of insect vectors when these
vectors feed
on an infected plant. When the insect vector moves to the next, healthy plant, the pathogens
detach and
infect the next plant. X. fastidiosa uses the same mechanism for its spread: it
attaches itself
to the chitin, before it forms a biofilm for further colonization of the insect.
As part of
our
Xylencer
phage, we want to equip the phage with a chitin binding protein to make sure it reaches all
infected
plants in a way that is less laborious to apply for farmers. To identify the ideal candidate
binding
protein, we did extensive literature research into different chitin binding proteins and tested
their
capacities.
Identifying candidates
The first candidate is the X. fastidiosa hypothetical protein PD1764. In a study conducted by Labroussaa et al., it was found that this hypothetical protein had chitin binding capabilities. When the LysM domain was knocked out of this protein, chitin binding was no longer possible [2].
The second candidate is LysM domain of PD1764. Since the adhesion protein will ultimately be placed on a phage capsid, a smaller domain could be favored. That is why the individual LysM domain is also tested individually.
A third candidate was the HAD-domain of the hemagglutinin-like protein of X. fastidiosa. The N-terminal domain of this protein also showed chitin binding capacities, but lost those capacities after knocking out the HAD-domain of this protein [2].
A fourth candidate is the 3LysM domain of the Cerk1 receptor of Arabidopsis thaliana. The receptor is used by the plant to detect the chitin of fungal cell walls and for resistance to fungal pathogens. The first 211 amino acids of the receptor will be tested, since this region contains the three LysM domains that are all necessary for the receptor to bind chitin [3].
The fifth potential candidate is the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. This domain was proven to be responsible for the binding of chitin and is available as a biobrick: BBa_T2028 [4].
The sixth potential candidate is truncated variant of the biobrick for the chitinase A1 domain. This version is 7 amino acids shorter than the biobrick variant, as it was described in literature [4]. This part was tested as well to have a good comparison between the two variants.
Protein Production
To test the chitin binding capacities of these proteins, they were produced using E. coli strain BL21DE3. This strain has a T7 RNA polymerase in its genome under an IPTG inducible promoter. After production, a pre- and a post-induction sample are loaded on a protein gel to check for recombinant protein production (Figure 2).
The pre-induction samples all have a similar band pattern; for PD1764 (lane 3, an extra band could be observed. In the post-inoculation sample, this protein band was much more abundant, indicating successful protein production of protein PD1764. However, a conclusion could not be given for the other proteins due to insufficient separation. The samples were loaded on a 12% acrylamide (Figure 4) and a 16% tricine gel (Figure 5 & 6). However, these gels also did not successfully separate the proteins. An alternative explanation could be that these proteins were not successfully folded and as a result, degraded. For Figures 4 to 6, see “Protein production problems".
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Protein production construct arrow_downward
The construct [5], used to produce the recombinant proteins (Figure 2) is submitted in the parts registry. It contains the following components:
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T7 promoter:
The strain, with which this construct is used is E. coli BL21DE3. This E. coli strain encodes the T7 RNA polymerase on its genome. To express this genomic encoded T7 RNA polymerase, the promoter upstream of it must be activated with IPTG.
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Bicistronic design (BCD):
Downstream of the promoter, a BCD-system has been placed. The mRNA encoded by this BCD contains two ribosome binding sites and encodes for a small peptide (approximately 16 amino acids). By transcription of the small peptide, the second ribosomal binding site (RBS2) becomes exposed due to the translation of the mRNA. This will remove any secondary RNA structures in the 5’UTR region of the protein of interest, which could have negative effects on translation efficiency. This way, protein production is will be increased [6].
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Strep-tag:
There is a strep-tag on the N-terminal end of the construct. By using a strep-tag, the recombinant protein can be isolated with both a gravity column or FLPC by running cell free extract over a streptavidin column and eluting with biotin.
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TEV cleavage site:
After the strep-tag, a TEV (Tobacco Etch Virus) protease cleavage site is encoded. When treating the recombinant protein with TEV protease, the strep-tag gets cleaved off the protein, resulting in pure recombinant protein.
Figure 3: Linear overview of the plasmid used for the production of recombinant protein. This is an example containing PD1764, for the other proteins they were inserted in its place. The expression cassette contains the T7 promoter, BCD design, strep-tag and TEV cleavage site. -
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Protein production problems arrow_downward
PD1764 could be produced without any troubles. However, the other protein (domains) were not visualized. This was also the case on a 12% acrylamide and a 16% tricine gel. An expected explanation for this could be that since the domains are not complete proteins, they do not get folded correctly and get subsequently degraded. To try to overcome this, an alternative approach was made. The construct was changed to Strep-tag → Maltose binding protein → TEV cleavage site → protein domain. First, the entire construct would be produced and then, after purification, the protein domain would be cleaved from the Maltose binding protein using TEV protease. After producing the construct, a size difference was visible. However, after cleaving the construct with TEV protease and loading again on a gel, the domain was not visible. This led us to abandon the production of the proteins via this route.
During our talks with iGEM Eindhoven about a potential collaboration we discussed this problem and they offered us to help troubleshooting this, since they also had experienced protein purification problems. They gave us three pieces of advice. Two of them, codon optimization of the gene and sonification, were already in our own protocols. They also advised us to lower the concentration of IPTG used for inducing the protein expression, since the IPTG might have toxic effects on the cell and therefore lower protein production. However, decreasing IPTG concentration did not yield higher protein production. More on the troubleshooting at the collaborations page.
Figure 4: Pre-induction vs post-induction for the adhesion protein candidates on a 12% acrylamide gel 1= 3LysM, expected size: 22.45 kDa. 2= LysM domain PD1764, expected size: 7.65 kDa. 3= PD1764, expected size: 44.52 kDa. 4= CBD ChitinaseA1, expected size: 7.91 kDa. 5= BBa_T2028, expected size: 7.25 kDa. 6= HAD domain, expected size: 14.77 kDa. (Weights are including Strep tag and TEV cleavage site) 
Figure 5: Pre-induction vs post-induction for the adhesion protein candidates on a 16% tricine+urea gel 1= 3LysM, expected size: 22.45 kDa. 2= LysM domain PD1764, expected size: 7.65 kDa. (Weights are including Strep tag and TEV cleavage site) 
Figure 6: Pre-induction vs post-induction for the adhesion protein candidates on a 16% tricine+urea gel 4= CBD ChitinaseA1, expected size: 7.91 kDa. 5= BBa_T2028, expected size: 7.25 kDa. 6= HAD domain, expected size: 14.77 kDa. (Weights are including Strep tag and TEV cleavage site)
Protein Purification
After successful protein production of PD1764, the next step was to purify the protein to perform binding assays with it. However, the protein was not obtained in the elution fraction after several attempts. To check for the protein in the membrane, the cell pellet obtained after cell lysis was loaded on a 12% acrylamide gel (Figure 7).
This indicated that the protein stayed in the pellet. A possible explanation for this was that the protein ended up in the membrane of the cell and therefore could not be purified. To test that hypothesis, a 3D structure was predicted for the protein, using the Phyre 2.0 tool [7]. This 3D structure was created by aligning the query protein to known amino acids and their 3D structure. Since there is not much known about this X. fastidiosa protein, only 41% of its amino acids were modelled with a certainty over 90% (Figure 8). The 3D structure was also predicted using the I-Tasser webserver [8-10] (Figure 9). Then, both structures were visualized using ChimeraX [11].
Both models differ quite a lot from each other, since the majority of the protein has no similarities to other proteins. However, both models indicated an alpha-helix (blue in Figure 8 and 9) in the beginning of the protein structure. To check whether this alpha-helix, or other helices in the protein structure are transmembrane regions, the entire protein construct, consisting of the strep-tag, TEV cleavage site and protein PD1764, was checked for transmembrane regions using the TMHMM program from DTU Bioinformatics [12].
The TMHMM result indicated that the first 20 amino acids are on the inside of the cell. The first 18 amino acids consist of the strep-tag and the TEV cleavage site. The 21st to the 40th amino acid of the total protein are predicted to be in the membrane. This region corresponds to the first 20 amino acids of the PD1764 protein. To overcome the problem of protein PD1764 ending up in the membrane, a new variant of this protein is made: PD1764sh, with sh standing for shorter. PD1764sh lacks the first 20 amino acids from the N-terminus. After verifying protein production using a 12% acrylamide gel, protein isolation was performed again. This time, the protein was successfully isolated (Figure 10).
Chitin Binding Assays
To test the chitin binding capacities of PD1764, chitin binding assays have been conducted. For this, Chitin Magnetic Beads from New England Biolabs were used. During the chitin magnetic bead assay, the flow through, wash fractions and elution fraction were saved and loaded on gel. However, the chitin binding was not successful with these beads, since no protein could be observed in the elution fractions. Besides this, there was also no difference in the intensity of the band of the sample and the flow through for both PD1764sh and BSA (negative control) (Figure 11).
To test for chitin binding, an alternative approach was used. PD1764sh was added to a chitin solution and incubated under shaking for an hour. Controls were PD1764sh without chitin and BSA with and without chitin. After incubation for an hour, the samples were spun down and the supernatant was collected. Then, this supernatant was tested on its protein concentration, using a Bradford assay. The hypothesis was that unbound protein stayed in solution, while bound protein remained in the pellet. Using a standard curve and the A595 of the samples (triplicates), the amount of protein in the supernatant was calculated. Then, the amount of protein bound to chitin was calculated, using protein samples incubated without chitin as control.
From this assay can be concluded that PD1764sh is able to bind chitin (Figure 12). Chitin binding could have been higher, if the assay was conducted with more chitin or if a longer incubation and centrifugation time was used.
The protocols used in these experiments can be found on the Protocols page under Protein Production, Protein Expression and Purification and Chitin Binding.
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References arrow_downward
- C. A. Durkin, T. Mock, and E. V. Armbrust, “Chitin in diatoms and its association with the cell wall.,” Eukaryot. Cell, vol. 8, no. 7, pp. 1038–50, Jul. 2009.
- F. Labroussaa, A. R. Zeilinger, and R. P. P. Almeida, “Blocking the Transmission of a Noncirculative Vector-Borne Plant Pathogenic Bacterium,” Mol. Plant-Microbe Interact., vol. 29, no. 7, pp. 535–544, Jul. 2016.
- E. K. Petutschnig, A. M. E. Jones, L. Serazetdinova, U. Lipka, and V. Lipka, “The Lysin Motif Receptor-like Kinase (LysM-RLK) CERK1 Is a Major Chitin-binding Protein in Arabidopsis thaliana and Subject to Chitin-induced Phosphorylation * □ S,” 2010.
- M. Hashimoto et al., “Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12.,” J. Bacteriol., vol. 182, no. 11, pp. 3045–54, Jun. 2000.
- Nieuwkoop, T., Claassens, N. J., & van der Oost, J. (2019). Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design. Microbial biotechnology, 12(1), 173-179.
- V. K. Mutalik et al., “Precise and reliable gene expression via standard transcription and translation initiation elements,” Nat. Methods, vol. 10, no. 4, pp. 354–360, Apr. 2013.
- L. A. Kelley, S. Mezulis, C. M. Yates, M. N. Wass, and M. J. E. Sternberg, “The Phyre2 web portal for protein modeling, prediction and analysis,” Nat. Protoc., vol. 10, no. 6, pp. 845–858, Jun. 2015.
- J Yang, R Yan, A Roy, D Xu, J Poisson, Y Zhang. The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12: 7-8 (2015).
- A Roy, A Kucukural, Y Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5: 725-738 (2010)
- Y Zhang. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, vol 9, 40 (2008).
- UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE. Protein Sci. 2018 Jan;27(1):14-25.
- A. Krogh, B. Larsson, G. von Heijne, and E. L. . Sonnhammer, “Predicting transmembrane protein topology with a hidden markov model: application to complete genomes,” J. Mol. Biol., vol. 305, no. 3, pp. 567–580, Jan. 2001.