Difference between revisions of "Team:Wageningen UR/Results/affinitybody"

Line 153: Line 153:
 
After several transformations an estimated library size of 110.000 affinity molecules was created. To check whether the library has a bias towards certain nucleotides and therefore amino acids, 96 colony PCR’s were performed on one of the transformations.   
 
After several transformations an estimated library size of 110.000 affinity molecules was created. To check whether the library has a bias towards certain nucleotides and therefore amino acids, 96 colony PCR’s were performed on one of the transformations.   
 
</p>
 
</p>
−
+
 +
<p>
 +
The PCR products were sent for sequencing and from 86 succesfull PCR reaction the data is depicted in Figure 5.
 +
</p>
 
<div class="figure-center">
 
<div class="figure-center">
−
<img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/5/5c/T--Wageningen_UR--Results_Tryps_SDS_rISG64.jpg"/>
+
<img class="figure-center-img" src="https://static.igem.org/mediawiki/2017/9/9d/Data_2.jpg"/>
 +
<div class="figure-center-caption">
 +
<b>Figure 3:</b> SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.  
 +
</div>
 
</div>
 
</div>
 
 

Revision as of 14:24, 25 October 2017

Affinity Molecule library

The goal of this project was making the library of affinity molecules to be used in the phage display. The modularity and specificity of the Mantis diagnostic system comes from the use of affinity molecules. These molecules are created by selecting them for their specificity via phage display from a random naive library. The approach taken in this project proved to be a reliable way to create such a library.



Introduction

Affinity molecules are antibody mimetics based on staphylococcal protein A (SPA) (Nord et al., 1997).The small, 6kDa, affinity proteins are based on the Z domain of the cell-wall anchored bacterial protein A. The native function of protein A is immunoglobin binding and contributes to evading the immune system (Nord et al., 1995). By making changes to 13 amino acids on 2 helices essential for specificity, affibodies for a wide variety of targets can be developed (Figure 1). Since its discovery affibodies have been developed for targets such as insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE and HER2 (Löfblom et al., 2010). Potential uses for these affibodies are imaging, purification, detection and many therapeutic applications (Löfblom et al., 2010).

Construct

The vector used to make the library with is pComb3XSS, acquired from AddGene. The pComb3XSS vector has an origin of replication for both e coli and filamentous phage M13. By using the SacI and SpeI restriction sites any protein of interest can be expressed as a fusion to the g3p protein. This protein is incorporated in the M13 helper phages upon infection of bacteria carrying this plasmid.

Figure 2: Amino acid sequence of wild-type IgG binding affinity molecule. Amino acid residues targeted for randomization in library creation in red.

The amino acid sequence of the wild-type IgG binding affinity molecule is depicted in Figure 2. The amino acids in red are the amino acid residues that are responsible for specific binding and will be targeted for randomization in the creation of the library.

Figure 2: Amino acid sequence of wild-type IgG binding affinity molecule. Amino acid residues targeted for randomization in library creation in red.

The Helix 3 region of the affinity molecule is not responsible for the binding specificity and will not be targeted for randomization. Therefore the Helix 3 region is ligated into the backbone before the library is integrated to make for an easier library ligation. The Helix 3 fragment was amplified with primers in such a way that it can be ligated into the pComb3XSS vector with the existing SacI/SpeI restriction sites. However a type-II restriction site (BsaI) was incorporated into the fragment to allow for the library integration without leaving a scar (Figure 3).

Figure 3: Overview of the Helix 3 fragment with SacI/SpeI restriction sites for cloning into the pComb3XSS vector. The internal BsaI restriction site allows for scarless integration of the Helix 1/Helix 2 library.

Oligo fragments were used to create the Helix 1 and Helix 2 fragments with random nucleotides on the desired places. The oligo’s are designed in such a way that there is a NN G/T degeneracy at the amino acid residues of interest. The NN G/T degeneracy improves the amount of non-sense codons produced by a NNN degeneracy and reduces the amount of stop codons as well. The annealed fragments for Helix 1 (top) and Helix 2 (bottom) can be seen in Figure 4.

Figure 4: Top: Fragment of annealed oligo's designed for Helix 1 with a NN G/T degeneracy. Bottom: Fragment of annealed oligo's designed for Helix 2 with a NN G/T degeneracy.

The Helix 1 and Helix 2 fragments were ligated into the linearized backbone (SacI/BsaI) and the ligation production were used for the transformation of XL1-Blue cells. The XL1-Blue cells have the following genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́proAB lacI qZ∆M15 Tn10 (Tetr)]. Important is that the strain used has a F pilus which is essential for the attachment of the M13 phages.

After several transformations an estimated library size of 110.000 affinity molecules was created. To check whether the library has a bias towards certain nucleotides and therefore amino acids, 96 colony PCR’s were performed on one of the transformations.

The PCR products were sent for sequencing and from 86 succesfull PCR reaction the data is depicted in Figure 5.

Figure 3: SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.
Figure 3: SDS gel of the protein fractions eluted from the strep-tactin column, both the flowthrough after loading the cell lysis onto the column, a few washing steps and the elution fractions.

The final 50 μl elution fraction (Elute 4) contains 283 μg/ml protein for rISG64, whereas the elution for rISG65 just contains 63 μg/ml protein. As seen from the high amounts of protein in the flowthrough, the column has reached its saturation point.

These tagged proteins, bound to the strep-tactin beads, are used for phage display selection.

Moreover, two biobricks were created of these constructs: BBa_K2387060 and BBa_K2387061. For this, the recombinant ISG gene, including the two tags, was cloned into the linearized pSB1C3 vector using biobrick assembly.

References

  1. Biéler, Sylvain, et al. "Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis." PloS one 11.12 (2016): e0168074.
  2. Sullivan, Lauren, et al. "Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device." PLoS neglected tropical diseases 7.2 (2013): e2087.
  3. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.