BLT Stages

Abstract Bachelor Project FBT 2019-2020: Validation of constructs with new potential recombinant cashew PR-10 allergens, A toolbox for identification of new cashew allergens

Purified individual allergens are used to provide insight into which allergens play a role in food allergies. In cashew nuts, only three purified individual allergens have been identified so far: Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin). A recent study has identified three novel proteins that show homology with pathogenesis related protein family 10 (PR10) proteins. The research aim is to demonstrate whether these proteins induce allergic reactions in patients with birch pollen associated cashew allergy, the proteins must be expressed in bacteria and purified. Therefore was decided to transform cashew nut constructs PR10-18220-11, PR10-25514-14 and PR10-25355-15 from an Escherichia coli (E.coli) BL21 (DE3) strain to E. coli BL21 (DE3) arginine / lysine strain, enabling the labelling of the allergens during induction. The constructs were validated after the transformation by demonstrating the presence of the allergens via a colony polymerase chain reaction (PCR), after which sanger sequencing was applied. Next, a selective growth medium (M9) was used to examine whether the constructs were placed in the correct bacterial strain. The protein expression was checked via SDS-PAGE. Finally, the purification of the recombinant histidine-tagged PR10-18220-11 fusion protein was optimized using cobalt beads affinity chromatography. All three constructs were found in the correct E.coli BL21 (DE3) arg-/lys- strain, both sequencing and protein expression showed that the correct proteins were present. Variation in imidazole concentration in the binding and wax steps showed that 5 mM imidazole gave the highest concentration of protein after elution with 250-500 mM imidazole. However, a large part of the protein remained bound to the beads. The three cashew nut constructs were transformed to the E.coli BL21(DE3) arg-/lys- strain and confirmed. The protein purification has to be optimized so that the PR10-proteins can be used in research to prove their allergenicity.

Abstract Bachelor Project 1 FBT 2018-2019: Cloning and characterization of new recombinant cashew allergens

Food allergy is a large problem in Western countries. Interestingly, there is still a lack of information regarding individual food allergens. For instance, only three cashew allergens have been identified so far and cashew Bet V 1-like allergens and oleosin allergens are not described yet. Recently using RNA-sequencing, five new possible cashew allergens have been detected and cloned in a pGEM-T vector. The aim of this study was to re-clone and characterize these five new recombinant cashew allergens into a pET16b expression vector. Re-cloning was performed using the Gibson assembly technique and all allergens gave a positive transformation result. To be sure that the sequences of all five allergens were correctly incorporated in the vector, clones were subjected to Sanger Sequencing. Furthermore, recombinant allergens were expressed and analyzed using LC-MS/MS. For the five clones, 100% homology with the original vector was found, but for all clones a minimum of peptides have been found with LC-MS/MS. After cloning, proteins were expressed and demonstrated with SDS-PAGE. All clones resulted in a protein that is specific for the allergen and these were used for LC-MS/MS. To identify these proteins as a potential source of food allergy, western blots with specific PR10 or oleosine antibodies were subsequently examined. Three out of five allergens gave cross-reaction with the antibodies.

Another experiment was performed using sera of two different volunteers, one positive and one negative sample for cashew allergy. Bet v 1 showed reactivity with the positive sample, like Pru av 1.0101, but showed a weaker affinity for the five cashew allergens. We therefore conclude that all allergens were successfully cloned and in one volunteer with pollen allergy there were found antibodies against the new recombinant cashew allergens.

Abstract Bachelor Project 2 FBT 2018-2019: CONVERSION OF GFP TO BFP WITH THE AID OF CRISPR-CAS9 GENOME EDITING

The CRISPR-Cas9 system is a multifunctional genome editing technology that operates in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 protein. The repair mechanisms of cells, make it possible to switch off specific genes by non-homologous end joining (NHEJ) or to replace a gene with a specific DNA sequence by homology directed repair (HDR). The ratio HDR/NHEJ is very low. To further investigate this ratio,a CRISPR-Cas9 model system was set up with K562 green fluorescent protein (GFP) positive cells. The purpose of this model system is to succesfully switch off GFP by NHEJ and converse GFP to blue fluorescent protein (BFP) by inserting a repair template by HDR. Two plasmid vectors were used, pX330 and pX459, to express the Cas9 protein but lack the sequence for the guide RNA. The plasmid were digested with BbSI and then ligated with the designed guide RNA insert. Transfection of K562-GFP cells with pX330 and pX459 constructs was performed by nucleofection, using a NEON Transfection System by ThemoFisher Scientific and a Nucleofector 2b device by Lonza. Ten days postnucleofection a maximum decrease of GFP was achieved (~40% using the Nucleofector 2b device using 1 000 000 cells and ~90% using the NEON Transfection System using 100 000 cells). Based on this knowledge another test was performed by using the HDR repair template. Likewise a maximum decrease of GFP was achieved ten days pos tnucleofection together with the development of a BFP positive population (~2% using the Nucleofector 2b device and 4% using the NEON Transfection System). These results proved that the use of the model system was succesfull. The GFP protein has been shut and at a low level been conversed to BFP through gene editing. This set up can be further optimised in the future to chance the NHEJ/HDR ratio to obtain a higher percentage of BFP positive cells.