Peptide deformylase from superbacteria

Transcription

1 Peptide deformylase from superbacteria

2 Antibiotics Most antibiotics were originally isolated from soil-derived actinomycetes between 1940s and 1960s (Golden era of antibiotic discovery) Natural product discovery became impractical owing to the increasing difficulty of identifying new classes of antibiotics against the background of known compounds.

3 Targets of antibiotics Nature reviews/ Drug discovery (May, 2013)

4 Antibiotics resistance and tolerance Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter faecium Nature reviews/ Drug discovery (May, 2013)

5 Introduction Super bacteria Top 6 dangerous super bacteria Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa Enterobacter Discover new drug (Xanthomonas oryzae pv. oryzae : Plant Pathogen)

6 Acinetobacter baumannii Acinetobacter baumannii is a Gram-negative round- or rod-shaped bacterium. Acinetobacter baumannii (Ab) is an opportunistic bacterial pathogen primarily associated with hospitalacquired infections. This means that it will only cause disease if you are already unwell; for example if you are immuno-compromised or are already taking antibiotics. Acinetobacter baumannii is responsible for 80% of all Acinetobacter infections There are in fact 34 species of Acinetobacter, most of which are actually innately resistant to antibiotics, rather than developing over time. In hospital outbreaks, it is considered as one of the most important nosocomial pathogens causing bacteremia, pneumonia, and other respiratory and urinary tract infections.

7 Antibacterial drug development Chan et al Drug Discovery Today

8 Pathogen whole genome sequences Target gene cloning or synthesis MIC by whole cell antibacterial screening IC50 by enzyme assay Structure-based drug design Fragment based drug design In silico screening

9 Introduction Peptide deformylase (PDF) PDF MAP Nature Reviews Genetics (2003) In bacteria, protein synthesis initiates with a formylated methionine. Pepide defromylase (PDF) is required to remove the formyl group from a newly synthesized polypeptide chain. This activity is essential for subsequent N-terminal processing by methionine aminopeptidase (Map).

10 Introduction Experiment procedure Protein purification Ammonium sulfate precipitation Affinity chromatography Ion-exchange chromatography Protein crystallization Structure determination Gene cloning Expression cell transformation CONCEPTS OF BIOLOGY(2015) Size-exclusion chromatography Protein expression optimization Purified protein Enzyme assay Enzyme kinetics Enzyme inhibition Structure analysis Enzyme reaction mechanism Protein-inhibitor interaction

11 Gene cloning Expression vector pet11a-n-ht N-terminal PaPDF_Ib, AbPDF_Ia, AbPDF_Ib 7 X His tag TEV cleavage site Protein * Tev cleavage site : Tobacco Etch Virus(TEV) protease recognition site pet29b-c-h Protein 6 X His tag C-terminal SaPDF_IIb pet11a XoPDF_Ib Protein Ab : Acinetobacter baumannii Pa : Pseudomonas aeruginosa Sa : Staphylococcus aureus Xo : Xanthomonas oryzae

12 Purification Protein purification strategy pet11a-n-ht 1 st Affinity chromatography 2 nd TEV cleavage step Last Ion exchange chromatography pet29b-c-h 1 st Affinity chromatography Last Ion exchange chromatography pet11a 1 st Ammonium sulfate precipitation 2 nd Size-exclusion chromatography Last Ion exchange chromatography

13 Achievements N-terminal 7 X His tag PaPDF_Ib pet11a-n-ht TEV cleavage site PaPDF_Ib

14 Expression Protein expression optimization Scale up Optimization 100 ml Induction time Expression temperature Inducer concentration 8 L Expression cell line

15 Expression Protein expression optimization : PaPDF_Ib (Size : 22.5kDa) kda M M 2H 4H 6H 8H 0H 2H 4H 6H 8H 0H 4H 8H 16H 0H 4H 8H 16H kda Insoluble part Soluble part Insoluble part 20 Soluble part Temperature : 37 IPTG concentration : 0.5mM Induction time : ~8H Temperature : 15 IPTG concentration : 0.5mM Induction time : 16H

16 Purification Purification procedure 1 st Affinity chromatography(ni-nta) 2 nd TEV cleavage step(ni-nta) Last Anion exchange chromatography Protein Yield : 173mg Protein Yield : 80mg Protein Yield : 73mg A B Fractions Rack Pos.: A Tube #: % Buffer B AU Min.Tenth ms/cm

17 Crystallization Protein crystallization Initial screening ~1,000 condition Optimization Hydra (Automated Pipetting Systems) 0.1 M Sodium Hepes; MOPS ph M CaCl M MgCl % (v/v) MPD 12.5% (w/v) P1k 12.5% (w/v) PEG 3, M Sodium Hepes; MOPS ph M CaCl M MgCl % (v/v) MPD 10.0% (w/v) P1k 10.0% (w/v) PEG 3,350

18 X-ray Crystallography X-ray diffraction and structure determination X-Ray Diffraction Molecular modeling Diffraction pattern Structure fitting to electron density map

19 Crystal structure 5 different crystals from 4 pathogens 2ul 10ul PaPDF_Ib AbPDF_Ia AbPDF_Ib SaPDF_IIb XoPDF_Ib

20 Crystal structure Inhibitor bound PDF structure Inhibitor

21 Achievements In vitro inhibition test