Optimizing Crystallization Conditions of the N-terminal Domain

Optimizing Crystallization Conditions of the N-terminal Domain of hAMPDA2 and Implementing a Baculovirus Expression System for hAMPD1 and 3 HisTrap Purification 50 47.5 mM Imidazole 1000 500 7 160 mM Imidazole 250 mM Imidazole -50 0 1 2 3 4 5 6 7 8 9 10 0 7 8 9 10 11 13 50 15 17 19 21 23 25 100 27 29 32 34 150 36 39 41 43 45 47 200 3 30 32 34 36 150 38 40 42 44 46 200 EcoRI-Flag-His-TEV-AMPD3-HindIII Flag-His-TEV-AMPD3-HindIII Ladder EcoRI-Flag-His-TEV-AMPD1-HindIII Flag-His-TEV-AMPD1-HindIII 2 2 Waste ml ml Figure 13. Agarose gels from insert steps. Figure 6. 2nd Purification Chromatogram Ladder Pooled concentrated fractions from first HisTrap purification Peak at 25% buffer B(fraction 32) Peak at 10% buffer B(fraction 25) Wash(fraction 13) Flow through(fraction 6 from FPLC) Supernatant Pellet Ladder Polyol pathway Cell Lysate Sucrose 3 5 49 51 Waste 250 Figure 5. 1st Purification Chromatogram Foods 5 7 Control(10 mg/mL N-term protein) 6 100 28 2nd HisTrap Peak at 100% Buffer B(f. 40) 5 24 26 2nd HisTrap Peak at 60% Buffer B(f. 35) 4 22 2nd HisTrap Peak at 60% Buffer B(f. 34) 3 50 16 18 20 1st HisTrap Peak at 100% Buffer B(f. 47) Fructose Metabolism For Hereditary Fructose Intolerance (HFI) 2 14 1st HisTrap Peak at 60% Buffer B(f. 39) 1 12 kb 7 5 3 2 -100 0 kb Ladder kb 0 81.25 mM Imidazole 47.5 mM Imidazole 81.25 mM Imidazole Flag-His-TEV-AMPD3-HindIII 100 His-TEV-AMPD3-HindIII 250 mM Imidazole TEV-AMPD3-HindIII 150 pET28a-hAMPD3 200 Flag-His-TEV-AMPD1-HindIII 1500 160 mM Imidazole His-TEV-AMPD1-HindIII 2000 mAU TEV-AMPD1-HindIII 2500 20190717 2nd HisTrap Nterm AMPD2 4 step AR001:10_UV1_280nm 20190717 2nd HisTrap Nterm AMPD2 4 step AR001:10_Conc 20190717 2nd HisTrap Nterm AMPD2 4 step AR001:10_Fractions pET28a-hAMPD1 mAU UNICORN 5.20 (Build 500) Result file: c:\...\Default\20190717 2nd HisTrap Nterm AMPD2 4 step AR001 20190716 HisTrap Nterm 1 AMPD2 4 step AR001:10_UV1_280nm 20190716 HisTrap Nterm 1 AMPD2 4 step AR001:10_Conc 20190716 HisTrap Nterm 1 AMPD2 4 step AR001:10_Fractions UNICORN 5.20 (Build 500) Result file: c:\...\Default\20190716 HisTrap Nterm 1 AMPD2 4 step AR001 Fructose is a simple ketonic monosaccharide that is found in numerous plants and is referred to as fruit sugar. Because high fructose corn syrup (HFCS) has a high sweetening power as well as long shelf-life, it quickly replaced sucrose as the primary sweeter in the diet. From 1970 to 2006, the sucrose consumption decreased from nearly 100g/day to close to 50 g/day while HFCS increased from close to 0g/day to nearly 60g/day. 1 In association with this significant increase in fructose consumption, hypertension, insulin resistance, and metabolic syndrome have increased.2 Fructose causes numerous complications in glucose metabolism with accumulation of triacylglycerol which alters the redox state that causes a systemic picture of insulin resistance leading to the development of obesity.3 Bacmid Generation Ladder Introduction Glucose kDa kDa 35 35 25 25 20 20 17 Figure 14. hAMPD3 in pFastBac1 sequencing. N-term hAMPDA 2 N-term hAMPDA 2 17 Mitochondrial dysfunction Figure 7. SDS-Page of purification steps and fractions from the 1st HisTrap purification Figure 1. Fructose Pathways Figure 8. SDS-Page of fractions from the 1st and 2nd HisTrap purifications Successive HisTrap purifications yield highly purified protein. The first chromatogram shows a high initial peak due to non-specific protein eluting off the nickel column. Fig. 7 shows that the target band (below 17kDa) is in the soluble fraction. Fig.7 demonstrates that some of the protein eluted off of the column at 47.5mM imidazole. This was not expected, as previous purification showed it did not elute until 160mM imidazole. Figure 6 and 8 shows non-specific protein initially present has been removed, with a clean band with only the target band present. Determining Protein Concentration Methods HisTrap Purification using FPLC Large-Scale Cell Growth in LB Media Sample from different points of Expression Process Run on 15% SDS-Page Gel μg N-term N-term (mg/mL) 2μL 20.84μg 10.42mg/mL Figure 9. Lowry assay BSA standard curve Cell Pellet with Protein Second HisTrap Purification using FPLC μL N-term Determining protein concentration using Lowry assay. N-term hAMPD2-2 does not have any tryptophans responsible for the absorption at 280nm. Using the Lowry assay to measure the absorbance at 750nm of oxidized peptide bonds, the concentration of protein was calculated using a standard curve from known BSA concentrations. Sample from Second Purification Run on 15% SDSPage Gel Crystal Analysis Apoenzyme Concentrated Protein from FPLC Concentration determined from Standard Curve from Lowry Assay Crystals set up using both hanging and sitting drop techniques Crystals Analyzed using LightMicroscope Figure 2. Expression, purification, characterization, and crystallization of the N-terminal domain of AMPD2 hAMPD1/3 PCR to add TEV Cleavage site and HindIII TEV HindIII PCR to add 6x HIS tag site HIS PCR to add FLAG tag site FLAG PCR to add EcoR1 site Figure 3. Gene Insert’s generation through successive PCRs pFastBac Transposition into Bacmid 9 % PEG 3350 1 % Tacsimate pH 5.0 Seed 100 10% PEG 3350 1 % Tacsimate pH 5.0 Seed 100 11 % PEG 3350 1 % Tacsimate pH 5.0 Seed 100 9 % PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 10% PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 11 % PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 9 % PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 10% PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 11 % PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 9 % PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 10% PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 11 % PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 9 % PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 10% PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 11 % PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 9 % PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 10% PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 11 % PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 9 % PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 10% PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 11 % PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 9 % PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 10% PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 11 % PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 Figure 11. Crystal Apoenzyme Enzyme + 1mM ATP EcoRI Ligation of Insert to pFastBac kb 7 5 1 2 Lane Primers Used Expected Band Length 1 Flag-His F and hAMPD3 R 2.5 kb 2 M13/pUC F and R 5 kb 3 AMPD2 is the liver isoform of AMPD that has been experimentally demonstrated in rabbit and mouse homologs to have harmful effects in regard to its hindering of AMP kinases (AMPK) 4, which improves insulin sensitivity in metabolic syndrome through decreased uric acid production. 5 AMPD has two additional isoforms AMPD1 and AMPD3 which are active in the skeletal muscle and brain respectively and are not related to fructose pathways in the liver. 5 While AMPD2 has been studied and characterized in various animal models, the full structure of the human protein is unknown, due to its rapid degradation during the purification process that often results in the truncation of the regulatory (N-terminal) domain.6 Based on previous experimentation that outlines the positive benefits of inhibiting AMPD2, this work looks to both characterize the structure of the regulatory domain of human AMPD2 and create bacmids for the AMPD1 and AMPD3 isoforms for insect cell expression. This will enable the creation of specific inhibitors that can isolate and target AMPD2 only, and these inhibitors can potentially be a treatment for diseases like metabolic syndrome. Cell Growth from DSMO Stock Ladder Transformation into E. coli NEB5! Growth of NEB5! Colonies on Agar plate Transformation into E. coli DH10bac Sequencing of DNA from selected colony to ensure insert ligated to pFastBac Figure 4. Bacmid generation for future baculovirus generation 9 % PEG 3350 1 % Tacsimate pH 5.0 Seed 100 10% PEG 3350 1 % Tacsimate pH 5.0 Seed 100 11 % PEG 3350 1 % Tacsimate pH 5.0 Seed 100 9 % PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 10% PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 11 % PEG 3350 2 % Tacsimate pH 5.0 Seed 10-1 9 % PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 10% PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 11 % PEG 3350 3 % Tacsimate pH 5.0 Seed 10-2 9 % PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 10% PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 11 % PEG 3350 4 % Tacsimate pH 5.0 Seed 10-3 9 % PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 10% PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 11 % PEG 3350 5 % Tacsimate pH 5.0 Seed 10-4 9 % PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 10% PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 11 % PEG 3350 6 % Tacsimate pH 5.0 Seed 10-5 9 % PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 10% PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 11 % PEG 3350 7 % Tacsimate pH 5.0 Seed 10-6 9 % PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 10% PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 11 % PEG 3350 8 % Tacsimate pH 5.0 Seed 10-7 Figure 12. Crystal with ATP Figure 10. Conditions screened for N-terminal hAMPDA2 domain crystal growth. Highlighted conditions correspond to crystal hits above. Only 2 wells produced one small crystal. 2 Figure 15. PCR amplification of hAMPD3 sequence in bacmid. Verification of hAMPD1 and hAMPD3 Gene Constructs. PCR samples from each insertion step were run on a 1% agarose gel. Target bands are 2404bp and 2494bp for hAMPD1 and hAMPD3, respectively. The gels display a clear band at approximately 2.5 kb after each PCR step indicating insertions were successfully added. Sequencing results confirmed successful ligation of hAMPD3 into pFastBac1 vector. Figure 15 confirms hAMPD3 insert in bacmid sequence with band at 5kb for bacmid and at 2.5 kb for AMPD3 gene alone. Conclusions • N-term hAMPD2 requires high protein concentration for crystallization. Fractions eluting at 47.5mM imidazole were not collected for 2nd purification. In addition, protein precipitated during the concentration process, resulting in further decrease in protein yield. • Seeding yields one microcrystal in both apoenzyme and enzyme with ATP conditions. Clear drops indicate protein did not precipitate and is well folded, but its concentration was not high enough for higher crystal yield. • Confirmed hAMPD3 insert in bacmid. The PCR generated insert was ligated into pFastBac1, which was transformed and transposed into a recombinant bacmid. • Unsuccessful ligation of AMPD1 into pFastBac1 Vector. This could be due to non-optimal annealing temperature. Future Work • Optimization of 4th PCR to add EcoRI site in hAMPD1. Future PCRs will be run to adjust the annealing temperature to add this site to the insert. • Generate Baculovirus of hAMPD3 for insect cell expression. These insect cell expressed protein will enable potential inhibitors to be tested to ensure they don’t inhibit the isoform better than hAMPD2 itself. • Further optimization of crystallization conditions to increase crystal yield and size. There needs to be further screening conditions around the hits from the tray. The crystals will then be analyzed using X-Ray crystallography for SAR. This will enable more potent inhibitors to be synthesized for future treatments targeting metabolic syndrome. • Optimization of purification conditions. In order to maximize protein yield during protein purification, the concentration of imidazole that the protein most readily elutes off will be further examined using SDS-Page protein gels to identify which concentrations contain the protein before the 2nd HisTrap purification.