fyp internal presentation (compiled)

Expression, purification and crystallization screening of

human Dicer helicaseEsther Tan Ding Qin (S10132125B)Monisha Joy Gomez (S10122011E)

Supervisor: Asst. Prof. Dahai Luo (LKC-SOM)Co-supervisor: Dr. Amy Ooi (NP) ss23

Contents

1. Introduction• Pathway• Domains, constructs• Issue

2. Materials & Methods• Flow- Mega primer design

3. Results• Heparin purification• TEV cleavage• Size-exclusion chromatography

3. Results• Crystallization• Thermoflour assay• RNAs• Buffers

4. Conclusion

5. Future work

Introduction (pathway)

stem-loop precursors→ microRNAsdsRNA→ siRNAs

Fragmented RNAs→ Gene silencing

(Yale, 2015)

1) Dicer2) TRBP3) Ago2

Introduction (domains, constructs)

• Various domains in Human Dicer helicase

• Helicase constructs D18 & D37 against full-length human Dicer

(Protein Production Platform, 2014)

Target protein present but significantly weaker than background

Target protein present and significantly stronger than background, Failed in purification

Introduction (issue)

• Erratic profile was observed • D37 eluted throughout

chromatogram

• D37 yield ↓ during concentration step.

Introduction (issue)

• Discovered structurally unpredicted region

• Replace region with short GSGS linker

SDM

SDM

Materials & Methods

Front RegionStructurally Unpredicted region

Back region GSGS Linker

Materials & Methods (mega primers)

Materials & Methods (flow)

Results (heparin purification)

Most proteins captured in main peak

Results (TEV cleavage)

15hrs TEV cleavage- Some protein still contained His6 tag.

22hrs TEV cleavage- Most proteins were His6 tag free.

Results (SEC chromatography)

• Good capture of protein within column volume

•Mutation allows proteins to be purified & concentrated

Results (crystallization)

Apo D37mpAfter 28 days,• Phase separation in droplets with

Crystal Screen I

• Optimizing conditions may increase crystal formation

Fraction Drop Condition (salt, buffer, precipitant)

SEC A 0.2M Ammonium acetate, 0.1M Sodium citrate tribasic dehydrate pH 5.6 30% w/v Polyethylene glycol 4,000

Heparin B 0.1 M TRIS hydrochloride pH 8.5, 8% w/v Polyethylene glycol 8,000

C 0.05 M Cesium chloride, 0.1 M MES monohydrate pH 6.5, 30% v/v Jeffamine M-600

D 0.2 M Magnesium chloride hexahydrate, 0.1 M TRIS pH 8.5, 3.4 M 1,6-Hexanediol

Results (crystallization)Droplets

Kit Clear Crystals Phase separation Precipitation

Crystal Screen I Few ×

Index ×

MIDAS ×

PEG/ Ion ×

SaltRx I ×

Protein concentration Protein concentration

Thermofluor assay

1. Protein is heated

2. Protein unfolds, revealing hydrophobic core

3. Fluorescent dye binds to core & emits signal

4. Fluorescent signal over temperature →

sigmoidal curveAddition of ligands may increase/ decrease

melting temperature depending on interaction

(Ray Salemme, 2013)

(Ray Salemme, 2013)

Results (Thermofluor- RNAs)

D37Tm increased 1-2°C when coupled to GG12, AG12 & SLA12

D37mpTm increased 7-15°C in RNAs• >45.0°C, ↑ crystallization (Dupeux,

2011)

• RNA binding ↑

dsRNA: GG12dsRNA: AG12ssRNA: SLA12synthetic analogue of dsRNA: poly(I:C)

D37 D37mp

Results (decreased Tm of mutant)

D37- 40.1°C• 422mM NaCl buffer

- Q-column fraction

• Bigger (348aa)

D37mp- 37.2°C• 300mM NaCl buffer

- SEC fraction

• Smaller (348- 48+ 4aa)• ↓ heat to unfold

SDM

>1 variable, reason for

decreased Tm inconclusive

Results (Thermofluor- salt buffers)

D37Tm increased slightly in HEPES pH 7.5 ONLY

D37mpTm increased 2-9°C across ALL salt buffers except BTP pH 8.5• pH increases, Tm decreases.

Same salt buffers are of one color, arranged according to increasing pH.

D37 D37mp

Conclusion

Proven structurally unpredicted region caused purification problems• Replacing region → purify & study part of helicase• Region removed interferes with RNA binding

• Optimize Crystal Screen I droplet conditions• Couple mutant with GG12, AG12, SLA12• Store mutant in BTP pH 6.3→ Crystals obtained will help establish human Dicer helicase structure

Future work

Introduce same mutation onto full-length Dicers of other species1. Run multiple sequence alignment on Dicer seq. of various species.

2. Identify least conserved structurally unpredicted regions.

3. Design mega primers to introduce mutation.

4. Obtain colonies with successful mutations.

Mus musculus Drosophila melanogaster Dcr1 Drosophila melanogaster Dcr2

Acknowledgements

Asst. Prof. Dahai LuoLee Kong Chian School of Medicine

Email: LUODAHAI@NTU.EDU.SGPhone: (+65)6586 9705

Office: Proteos #07-03B, Biopolis

Dr. Ming Wei ChenLee Kong Chian School of Medicine

Dr. Amy OoiNgee Ann Polytechnic

We also deeply appreciate assistance from Dr. Chong Wai Liew, Hui Yee Yong, Kouhan Li, Wint Wint Phoo, Yee Hwa Wong & Yongqian Zhao, which

has helped us along the way.

References

• Salemme, R. (2013). B1. Label Free Drug Screening. [online] Available at: http://www.beta-sheet.org/page11/page17/page18/index.html [Accessed 2 Feb. 2015].

• Salemme, R. (2013). E. Thermofluor Updates. [online] Available at: http://www.beta-sheet.org/page11/page45/index.html [Accessed 2 Feb. 2015].

• Dupeux, F., M. Rower, G. Seroul, D. Blot and J.A Marquez. (2011) A thermal stability assay can help to estimate the crystallization likelihood of biological samples. CrossMark, D67, 915-919

• n.a., (2015). Hampton Research. [online] Available at: http://hamptonresearch.com/tip_detail.aspx?id=150 [Accessed 16 Jan. 2015].

• Soding, J., A. Biegert and A.N. Lupas. (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res, 33, W244-248.

• Wostenberg, C., J.W. Lary, D. Sahu, R. Acevedo, K.A. Quarles, J.L. Cole and S.A. Showalter. (2012) The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs. PLOS ONE, 7 (12), 1-12.

• Yale.edu, (2015). [online] Available at: http://www.yale.edu/giraldezlab/miRNA_files/shapeimage_4.png [Accessed 3 Feb. 2015].