Team:Aboa/Results
Results
Our project aimed for the creation of a novel immunoassay that uses expanded genetic code to introduce a reactive azide moiety site specifically to Fabs that can then be forced to adopt a correct orientation on a biosensor surface. This would improve the repeatability and sensitivity of any assay that currently uses a random immobilization of antibodies.
Characterization of E.coli 321.A
As C321.ΔA.exp is the first genomically recoded organism (GRO) with interesting properties such as the inability to terminate TAG codons as it has had Release Factor 1 (RF1) completely removed (Lajoie et al. 2013). This is why we wanted to characterize this strain and see what sort of properties it has together with plasmid pEVOL-pAzF in producing a protein that has a site for incorporation of unnatural amino acids.
This experiment was performed using a 96-well plate and Cytation 5 multimode fluorescence imager. First the C321.ΔA.exp containing pEVOL-pAzF and pUC19[GFP-TAG-RFP] were precultured in the wells at 37°C in vigorous shaking and their OD600 was measured until it reached an average of 0.5. After that the wells were induced with varying amounts of Arabinose, IPTG and pAzF (Figure 1). The fluorescence was measured every 30 minutes at manufacturer preset GFP and mCherry channels. The results were normalized by reporting a relative change of RFP and GFP ratio.
Figure 1. The relative change of fluorescence measured in preset mCherry and GFP channels. pAzF has the strongest effect on the expression of RFP when the synthetase is present. This also shows that when the synthetase is not present, there still is some misincorporation of other amino acids to the TAG site as it cannot be terminated either. IPTG does not have an effect on the reading of TAG stop codon as expected.
Producing Anti-Digoxigenin Fab
We used a digoxigenin binding Fab fragment as our model antibody in this project. First of all this Fab has been vigorously optimized by codon harmonization to reduce its toxicity and to increase its yields as we were not sure how well this new strain could tolerate these humanized and toxic proteins (Kulmala et al. 2017). The second reason for using this was that the analyte, digoxigenin was readily available for us at the Department of Biotechnology as a labeled and non-labeled version. Although we later found out thet the Eu3+ labeling by the department chemists was not successful so we had to use other means to complete our measurements.
We started the production phase with an optimization task to find out what were effects of Fab variants to our cell viability and also to optimize the amount of IPTG, Arabinose and pAzF to use in an attempt to conserve expensive reagents. We were successful in producing three variants of the Fab fragment: Fab0 (unmodified Fab), Lys 108 (Lys->TAG, 108 representing the site) and Lys 191. We also made vector that contained a C-terminal TAGTAA modification after a 6xHis tag that we used together with the unmodified Fab. The histidine tag is directly opposite to the binding part so we used this as the binder that forces a correct orientation for the antibodies.
Figure 2. The end densities of the cells after a test production round. A small effect is already observed with these seemingly similar versions of the same protein.
Figure 3. Production optimization of Fab variants. The cells were tested (A and B) without pAzF present (C and D) and 1 mM pAzF present. (A and C) Concentration of Arabinose was tested and (B and D) concentration of IPTG was tested. From here we can observe that pAzF has the most dramatic effect in expression of the Fab when the synthetase is present. IPTG has a relative small, if any effect on the expression which is somewhat surpricing. We chose to use 1mM pAzF, 0,5 mM IPTG and 0,5 % Arabinose in our work. This also proved that we can produce Fabs using our GRO strain.
Figure 4. (A) 1µg of Ni-NTA purified pAzF modified and unmodified Fabs. (B) SEC purified unmodified Fab. (C) The labeling of our produced Fab with DBCO-Cy5.5 visualized using Li-COR Odyssey at 700 nm bandwidth. A clear labeling of the C-terminal pAzF (amino acid residue 460) can be observed from this picture. No unspecific labeling of Fab0 can be observed. Copper free click-chemistry displays a very specific way of labeling irrespective of the purity of the sample. The other variants (191, 108) were not labeled very efficiently. This could be due to poor solvent accessibility of the pAzF residue or degradation of the protein. Unbound label can be seen at 1 kDa.
Producing Anti-Dengue Fab
We were not able to successfully produce any dengue binding Fab fragments. This Fab sequence proved out to very toxic for our cell lines even in XL-1 Blue. Using IDT gBlocks as a synthesis method for these Fabs is not recommended as most of the genes we were able to sequence, proved out to be either truncated proteins or frameshifted. This also shows that the viability of those cells that harbor the correct gene was very low. After a huge sequencing effort we were able to find one unmodified fragment that we produced but we could not measure any specific Fab activity using Eu labeled 2A11 anti Fab antibodies.
Coating and testing Anti-Digox Fab
This part was what we were aiming for during the whole project. Because there wasn’t any DBCO modified 96-well plates available anywhere, we decided to use DBCO-coated magnetic beads (Jena, Biosciences, Germany). These beads could be coated with our pAzF modified Fabs. We first had to prove that the Fabs are able to bind to the beads specifically via the pAzF and not passively or in any other way.
We used two different approaches to immobilize the Fabs. In the first one, we added biotin group to the azide group by using biotin-DBCO, after which the Fabs were immobilized on a streptavidin-avidin plate (SA-plate) to react with the biotin. In the second approach, the Fabs were directly immobilized onto DBCO-magnetic beads. With Eu-2A11 in both the approaches we observed, that the azide group in pAzF can be used to immobilize the Fabs (Figure 5).
Figure 5. Determining the immobilization of Fabs via their pAzF onto a SA-plate (A) and onto DBCO magnetic beads (B). According to the fluorescent measured, there is more randomly immobilized Fab in the SA-plates compared to c-terminal conjugation. However, on the magnetic beads the situation is the opposite. Also, the signal from the Fab 191, Fab 108 and Fab0 are near the same level.
Because we did not have Eu-labeled digoxigenin available, we resorted in using Cy5-labeled digoxigenin kindly provided to us by our advisor Tuomas Huovinen. We then proceeded to measure these coated beads in a flow cytometer.
Figure 6. (A) The size (Forward scatter, FSC-area) and shape (Side scatter, SSC-area) distribution of randomly and site-specifically immobilized beads. This shows how a site specifically labeled antibody with only one reactive site does not cross-link the beads in the same manner as a randomly conjugated antibody. (B) A histogram of fluorescence intensity of Cy5 at 700nm. the total population 100000 beads. Uncoated beads in red. Passively adsorbed beads in blue, random conjugation in orange and site specific conjugation using pAzF in green. A much larger variance is achieved when using random conjugation because of the crosslinking of beads. Site specifically coated beads are more uniform. (C) when monomeric beads are measured, a much more uniform coating is once more observed with virtually no unspecific binding or empty beads with a huge difference in variance. Variance of the assay is a critical factor for the sensitivity and repeatability of any test.
Figure 7. A) and B) show the assay performance as signal to background ratio and the error as a robust coefficent of variation. The test error is already, without any optimization at a clinically relevant level
Temperature stability measurements of Fab fragments
Melting temperature of Fabs was performed with Prometheus NanoDSF differential scanning fluorometer (Munich, Germany). These measurements were kindly provided to us by our sponsor PerkinElmer Turku.
Figure 8. (A) Melting temperatures of Fabs. (B) Shown as the proteins change of fluorescence at 330 nm. The temperature stability of Fab with a pAzF at the histag does not alter the stability of Fabs.