Medicinal Structural Genomics of Parasitic Protozoa (MSGPP) Protein Production Pipeline

1. Medicinal Structural Genomics of Parasitic Protozoa (MSGPP) Protein Production Pipeline Alberto J. Napuli, Ph.D. MSGPP Protein Production Unit University of Washington, Biochemistry Seattle, WA

2. Medicinal Structural Genomics of Parasitic Protozoa (MSGPP) • April 2006 NIH funded project to determine crystal structures of medically relevant proteins of pathogenic protozoa. Goal is 50 structures and 250 ligand bound. • MSGPP is composed of 5 units • Target Selection • Protein Expression and Purification • Crystal Growth Unit • Structure Determination • Ligand Screening • Protein Expression and Purification • Cloning, Screening and Protein Purification • Recombinant protein expression in E.coli • Ligation Independent Cloning • Auto-induction • 4 purifications per week (~200/year) • Bottlenecks • Expression of soluble protozoa proteins in E.coli • Success rate from cloning to purification ~ 7% • Generate ~200 clones per month • Compact Protein Domain Designed w/large C and N terminal deletions • Multi-Species Game • 2008 Nibbles • Seattle Structural Genomic Center of Infections diseases (SSGCID) • Success rate from cloning to purification ~60 -80% • 4 purifications per week (~200/Year)

3. AVA0421 3C MAHHHHHHMGTLEAQTQGPGS- ORF GPGS- ORF MAHHHHHHM- ORF AVA0421 Only 21.85% AVA0421+ BG1861 50.33% BG1861 Only 27.81% Expression vectors BG1861 BG1861 AVA0421 Cloning Technique LIC LIC Antibiotic Resistance Amp Amp Promoter T7 T7 N-Terminal Fusion His His-3C Termination Codon TAA TAA

4. LIC Ready Vector (AVA0421) 3C site NruI PmeI atg gct cat cac cat cac cat cat atg ggt acc ctg gaa gct cag acc cag ggt cct ggt tcg cga ata ttc cta gct ttg ttt aaa cag cac gaa caa gtt tac cga gta gtg gtc gtc gtc gtc tac cca tgg gac ctt cga gtc tgg gtc cca gga cca agc gct tat aag gat cga aac aaa ttt gtc gtg ctt gtt caa RESTRICTION REACTION 3C cleavage site atg gct cat cac cat cac cat cat atg ggt acc ctg gaa gct cag acc cag ggt cct ggt tcg tac cga gta gtg gtc gtc gtc gtc tac cca tgg gac ctt cga gtc tgg gtc cca gga cca agc aaa cag cac gaa caa gtt ttt gtc gtg ctt gtt caa T4 DNA polymerase activity atg gct cat cac cat cac cat cat atg ggt acc ctg gaa gct cag acc cA tac cga gta gtg gtc gtc gtc gtc tac cca tgg gac ctt cga gtc tgg gtc cca gga cca agc dATP only 5’ aaa cag cac gaa caa gtt Aa 3’ to 5’ exonuclease activity PCR Insert g ggt cct ggt tcg atg-orf-xxxxxxxxxxxxxxxxxx-T Tag-orf-xxxxxxxxxxxxxxxxxx-a ttt gtc gtg ctt gtt c g ggt cct ggt tcg ATG-ORF-xxxxxxxxxxxxxxxxxx-TAA TAA cag cac gaa caa g c cca gga cca agc tac-orf-xxxxxxxxxxxxxxxxxx-att att gtc gtg ctt gtt c dTTP only T4 DNA polymerase

5. PCR and Ligation Independent Cloning DNA templates Genomic and cDNA PCR Gel Extraction Prep BG1861 & AVA0421 LIC Ready Exp. Vectors REV Primers T4 DNAP Exonuclease Reaction FWD Primers PCR Master Mix Ligation Independent Cloning LIC Transformation NovaBlue

6. Colony PCR NovaBlue Colonies X2 H2O Master Plate LB-AMP 37C 16 hr H2O REV Primers FWD Primers PCR Master Mix Plasmid Prep Plasmid DNA Clones

7. HT Screens 3 ml 20C PA0.5G 16 hr 3 ml 20C ZY-5052 16 hr Transformation BL21(DE3) 37C LB-AMP 16 hr Plasmid DNA Glycerol Stock Room Temp 40 min Lyses Buffer w/ 0.5% CHAPS+ Benzonase -80C Total/Expression Wash x3 Ni2+ Beads Elution Buffer 500 mM Imidazole Soluble Fraction Wash Buffer 30 mM Imidazole Waste Pure/Soluble

8. SDS/PAGE HT Screens-Analyses Total/Expression Pure/Soluble SDS/PAGE Samples 90C / 5 min

9. 4000 ml Flask 500 ml Flask 12 ml Falcon Tubes 100 ml 20C PA0.5G 200 rpm 16 hr 3 ml 37C LB-AMP 16 hr AIR 30 ml 10 colonies Transformation 500 ml Flask 37C LB-AMP 16 hr 100 ml 20C LB-AMP 200 rpm 16 hr 2000 ml 20C ZY5052 24-48 hr Large Scale Expressions - Upscales 1 ml 0.5 ml 0.1 ml 1000 ml 20C ZY5052 200 rpm 48-72 hr 1 ml Glycerol Stock PA0.5G AIR vs Shaking Glycerol Stocks Vs Fresh Colonies PA0.5G vs LB Plasmid DNA

10. Large Scale Expression Innova 44R x6 Capacity 6X1L ea / 36L total 2L per upscale = 18 targets per week LEX 48 Bioreactor x1 Capacity 48L total: 48X1L or 24X2L cultures 2L per upscale = 24 targets per week

11. Large Scale Expressions - Harvest 1L X2 10 ml 1 ml Sequencing 10 ml Screen 20-30 Grams -80C

12. Purification Schema

13. Lyses Buffer 200 ml 30 min 50% duty Ice Bath Sonication Benzonase 250 ml Room Temp 45 min Large Scale Lyses Lyses Buffer Lysozyme 25 ml 30C water Bath 15 min 1 hr 14,000 rpm 4C 20 mM HEPES pH 7.0 30 mM Imidazole 500 mM NaCl 0.5% CHAPS 1 mM AEBSF 5% glycerol 1 mM TCEP Lysozyme X8 Clarified Sample

14. Ni2+ Chromatography Explorer 100 X2 Ni2+ Column: 5 ml HisTrap FF Equilibration/Wash Buffer: 20 mM HEPES pH 7.0, 500 mM NaCl, 30 mM imidazole, 5% glycerol, 1 mM TCEP Elution Buffer: 250 mM imidazole. Sample Volume: 200-250 ml Sample Pump Flow Rate: 5 ml/min System Pump Flow Rate: 5 ml/min Equilibration: 10 CV Wash: 20 CV Elute: 10 CV Fraction Size: 5 ml Total Fractions: 12

15. 3C-protease cleavage reaction 3C Protease 1:50 20 mM HEPES pH 7.0 60 mM Imidazole 800 mM NaCl 5% glycerol Dialyses 4C/16 hr 20 mM HEPES pH 7.0 30 mM Imidazole 500 mM NaCl 5% glycerol Concentration: 3-20 mg/ml Total protein: 30-200 mg Volume: 6-20 ml 20 mM HEPES pH 7.0 200 mM NaCl 5% glycerol 1 mM DTT 20 mM HEPES pH 7.0 250 mM Imidazole 500 mM NaCl 5% glycerol 1 mM TCEP Ni2+ Ni2+ FT 15-40 ml W 10 ml E 10 ml

16. X2 SEC Chromatography Column: 300 ml 26/60 Superdex 75 SEC Buffer: 20 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 1 mM TCEP Sample Volume: 6-12 ml Sample Pump Flow Rate: 0.5-1 ml/min System Pump Flow Rate: 0.5-1 ml/min Equilibration: 0.5 CV Elution: 0.7 CV Fraction Size: 5 ml Total Fractions: 47

17. HPLC system and column maintenance

18. Protein Concentration 20 to 2 ml concentrate Pre-Crystalyzation Test PCT Dilute 96 Well PCR Plate 20-50 ul / well Flash Freeze Store at -80C

19. Supported by Supported by Supported by NIAID NIAID NIAID Acknowledgements Wim Hol Wesley Van Voorhis Christophe Verlinde Fred Buckner Ethan Merritt Erkang Fan Structure Determination Unit Isolde Le Trong Jüergen Bosch Jon Caruthers Eric Larson Crystallization Growth Unit Brian Krumm Jenni Ross Helen Neely Liren Xiao Li Zhang Protein, Expression & Purification Alberto Napuli Angela M Kelley Natascha Mueller Lisa Castaneda Soraya Aalami Selma Alkafeef Hauptman-Woodward MRI George DeTitta Joseph Luft Angela Lauricella Ligand Synthesis & Screening Sayaka Shibata Zhongsheng Zhang Informatics and Databases Frank Zucker Christine Stewart Supported by NIAID.