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Large Scale Protein Production Platform in TB Structural Genomics

Large Scale Protein Production Platform in TB Structural Genomics. Large Scale Protein Production Protocol. Fermentation. His-tag affinity (IMAC) purification. Gel filtration. or. Failed product (for each step). Ion-exchange (Bio-Cad 700E). Recycle. Concentration.

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Large Scale Protein Production Platform in TB Structural Genomics

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  1. Large Scale Protein Production Platform in TB Structural Genomics

  2. Large Scale Protein Production Protocol Fermentation His-tag affinity (IMAC) purification Gel filtration or Failed product (for each step) Ion-exchange (Bio-Cad 700E) Recycle Concentration

  3. Goals for Protein Production Facility For each protein, Concentration : >10 mg/ml Purity : > 95 % Volume : 1 - 5 ml Amount : >10 mg - 4x proteins per week - 1x column/analysis per day

  4. Flow chart for IMAC condition scouting Flow-rate Measurement Column capacity determination Vacuum manifold system design Elution condition scouting Resin reusable number determination Minimum elution volume determination Required solution volume determination Solution bottle size determination Overall running time determination

  5. Flow chart for Running Condition Scouting for Superdex 75 (26mm D x 600mm L)Gel Filtration Column GF column resolution study Extra part items determination GF column capacity test Maintenance item/schedule determination Sample loading volume study Solution bottle size determination & instrument space design Flow-rate study Overall running time determination Required solution volume determination

  6. AU 0.1 - Blue Dextran 2,000 kDa BSA 66 kDa Cytochrome C 12.4 kDa Carbonic Anhydrase 29 kDa 2 hours 0.5 hour 1 hour

  7. GFC capacity and loading volume test 1.0 AU 0.5 AU V = 1 ml V =2.5 ml Sample (V x 0.5mg/ml Aprotinin + 5mg/ml Carbonic Anhydrase + 0.5mg/ml BSA) 0.5hr 1 hr 2 hr 0.5hr 1 hr 2 hr 0.5hr 1 hr 2 hr 0.5hr 1 hr 2 hr 2.0 AU 2.0 AU V = 5 ml V = 10 ml

  8. Results of sample loading volume test for Superdex 75 (26mm D x 600mm L) GF column Velution/Vo Loading volumn of Sample Mixture 0.5mg/ml BSA 5mg/ml CA 0.5mg/ml Aprotinin 1 ml 1.29 1.65 2.57 2.5 ml 1.31 1.66 2.56 5 ml 1.32 1.67 2.56 10 ml 1.35 1.69 2.52

  9. Summary of Test for Superdex 75 (26mm D x 600mm L) GF column Resolution : good (within MW: 6,000 – 70,000 Da) Capacity : up to 50 mg (one sample), 60 mg (with 10 % of two contaminants) Loading volume : up to 10 ml Flow-rate : 3 ml/min (with run-time, 2.5 hrs)  Remained task : setting up parallel system

  10. Flow chart for Ion Exchange Column Resin & Condition Scouting with BioCad Workstation 700E pH scout Flow-rate study Gradient adjustment Sample autoloader function check Spare part item determination Running time minimization for each step Column capacity determination Solution bottle size determination & instrument space design Maintenance item / schedule determination Overall Running time determination Required solution volume determination

  11. pH scout with HS/M Cation Exchange column (4.6 mm D x 50 mm L) Sample : 50 ul of Ribonuclease A (4mg/ml, pI: 8.9) + Lysozyme (1 mg/ml, pI: 11) flow-rate : 10 ml/min pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH scout with HQ/M Anion Exchange column (4.6 mm D x 50 mm L) Sample : 50 ul of Ovalbumin (5 mg/ml, pI: 5.2) + BSA (5 mg/ml, pI: 4.7) flow-rate : 10 ml/min pH 6.5 pH 7.5 pH 8.5 pH 9.5 pH 5.5

  12. Summary of Poros Ion Exchange Column Test with BioCad Workstation 700E CationAnion 4.6Dx50mmL 10Dx100mmL 4.6Dx50mmL 10Dx100mmL Capacity 20mg 100 mg 20 mg 100mg pH 4, 5, 6, 7, 8 5.5, 6.5, 7.5, 8.5, 9.5 Sample volume 300 ul 10 ml 300 ul 10 ml Flow-rate 5 ml/min 20 ml/min 5 ml/min 20 ml/min Run-time 10.31 min x 5 23.27 min 10.31 min x 5 23.27 min Gradient (salt) 0.1 – 1.0 M 0.1 – 1.0 M (select from pH scout) (select from pH scout) per run (adjust based on pH scout ) (adjust based on pH scout ) Solution volume 74 ml x5 470ml 74 ml x 5 470 ml (total per run)

  13. Rv3465(dTDP-4-dehydrohamnose 3, 5-epimerase) Rv1299 (pI : 4.94)(peptide chain release factor 1) Fermentation IMAC Fermentation IMAC GFC GFC Anion Ex col : pH scout  7.5 Large scale Anion Ex col at scouted pH 7.5 Concentration Concentration (26.93 mg/ml) (12.06 mg/ml)

  14. Final Concentrated Proteins Sent for Crystallization Trials Rv0046c Rv2238c Rv3465 Rv1412 Rv3110 Rv2225 Rv2607 Rv2498c Rv2150c Rv2359 Rv2031c Rv3841 Rv1606 Rv1848 Rv3119 Rv2593c Rv3323c Rv3275c Rv2925c Rv1299 Rv1909c Rv2428 Rv1611 Rv2204c Rv3420c Rv0733 Rv2523c Rv2461c Rv3277 Rv0991c Rv2703 Rv2632c Rv2302 Rv0298 Rv2283 Rv2377c Rv0340 Rv2697c Rv3418c Rv1340 Rv3321c Rv0108c Rv0028 Rv0543c Rv2304c Rv1893

  15. Summary of Large Scale TB Protein Purification Purification trial : 91 Purified proteins : 46 Proteins on recycle list : 45 Protein #Reasons Suggestions 15 low binding on IMAC two-tag 14 low solubility evolution, re-folding 7low expression re-cloning 4 contamination after all two-tag / affinity col 3 low yield after all large scale culture2 precipitation evolution, re-folding

  16. TB Structural Genomics Consortium Leader : Tom Terwilliger TB Structural Genomics Protein Production Team members Min S, Park Chang Yub, Kim Beomseop, Rho Becky, Beccam Uhnsoo, Cho Team leader (assignment) Supervising overall protein production facility operation (assignment) Protocol and method development, research and data analysis, Ion Ex column operation and final product quality control and shipping (assignment) Protocol and method development, research and data analysis, quality control by DLS analysis (assignment) His-tag resin binding assay, fermentation, research and data analysis, administrating SGPP facility grand chart. (assignent) IMAC and GFC operation, concentration, analysis (MS and gel) and quality control, administrating TB SGPP facility online data. TB Structural Genomics Cloning Team members

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