Characterization of Dynein Solubility using Small Ubiquitinlike Modifiers'

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Characterization of Dynein Solubility using Small
Ubiquitin-like Modifiers.

• Brian Phan
• Dr. Elisar Barbar
• HHMI
• Summer 2009

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Introduction
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• pH levels
• Solvent
• Temperature
• Concentration
• Time

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Microtubule
ATP
Converts chemical energy from ATP into mechanical
energy.
Transports cellular cargo along microtubules.
Cellular Cargo
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Relevance

• Structural and mechanistic dysfunction in dynein
  can provide understanding to diseases and
  disorders.
• Provide insight into producing pharmaceutical
  agents that prevent aggregation.
• Leads into further studies for protein chemistry.

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Background (cont.)
Small ubiquitin-like modifier (SUMO). Protein
modifier which attaches itself to other protein
substrates. Discovered in 1996. Helps protein
regulate cellular processes.
http//www.mskcc.org/mskcc/_assets/content-image/2
15901.jpg
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Hypothesis SUMO helps the dynein intermediate
chain become more soluble in solution.
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Methods

• Grow and purify SUMO-protease.
• Use SUMO protease to cleave off SUMO from ICdel
• Use gel to identify if enzyme is active in
  cleaving off SUMO

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Results
Affinity Buffer Content 20mM Na-Phosphate 500mM
NaCl 5mM B-mercaptoethanol 5 Glycerol 10mM
Imidazole 1mM Na-azide

• SDS Gel of SUMO-protease after Affinity
  purification.
• Molecular Marker
• Flowthrough
• Wash
• 50mM Imidazole
• 350mM Imidazole

MW 55kDa 43kDa 34kDa 26kDa 17kDa
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Affinity Buffer Content 20mM Na-Phosphate 500mM
NaCl 5mM B-mercaptoethanol 5 Glycerol 10mM
Imidazole 1mM Na-azide pH9.0

• SEC Chromatogram for SUMO protease where Abs280
  nm is plotted against elution time on 45 mL SEC
  Column. Buffer used was common affinity buffer.

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• SEC Chromatogram for SUMO protease where Abs280
  nm is plotted against elution time on 90 mL SEC
  Column.

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Aff. Buffer Content 50mM Tris-Base 350mM
NaCl 10mM Imidazole 1mM B-mercaptoethanol 20
sucrose pH8.0

• SEC Chromatogram for SUMO protease where Abs280
  nm is plotted against elution time on 90 mL SEC
  Column. Buffer used was prescribed by Structural
  Biology Program at Cornell University.

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Aff. Buffer Content 50mM Tris-Base 350mM
NaCl 10mM Imidazole 1mM B-mercaptoethanol No
sucrose pH9.0

• SEC Chromatogram for SUMO protease where Abs280
  nm is plotted against elution time on 90mL SEC
  Column. Buffer used was prescribed by Structural
  Biology Program at Cornell University.

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Affinity Buffer Content 20mM Na-Phosphate 500mM
NaCl 5 Glycerol 10mM Imidazole 1mM
Na-azide pH9.0

• SEC Chromatogram for SUMO protease where Abs280
  nm is plotted against elution time on 90 mL SEC
  Column. Buffer used was original buffer.

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Results
MW 43kDa 34kDa 26kDa 17kDa
1 hr at 30oC
SUMO-CAT 39kDa CAT 25kDa SUMO 15kDa
Lane 1 Molecular Marker Lane 2 1500 Lane 3
11000 Lane 4 13000 Lane 5 15000
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Results
MW 55kDa 43kDa 34kDa 26kDa 17kDa
24 hours at 4oC
SUMO-CAT 39kDa CAT 25kDa SUMO 15kDa
Lane 1 Molecular Marker Lane 2 1500 Lane 3
11000 Lane 4 13000 Lane 5 15000
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Results
MW 43kDa 34kDa 26kDa 17kDa
48 hours at 4oC
SUMO-CAT 39kDa CAT 25kDa SUMO 15kDa
Lane 1 Molecular Marker Lane 2 1500 Lane 3
11000 Lane 4 13000 Lane 5 15000
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Conclusions

• Use of sucrose leads to possible protein
  aggregation during purification.
• Higher pH levels change protein solubility.
• SUMO-protease is active in cleaving off SUMO from
  substrate.

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Future Research

• Grow and purify an ICdel (92-261) construct.
• Grow and purify a SUMO ICdel( 92-261) construct.
• Analyze solubility levels of ICdel modified by
  SUMO and solubility levels of ICdel not modified
  by SUMO.

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Acknowledgements

• Dr. Elisar Barbar
• Dr. Kevin Ahern
• Barbar Lab Staff
• Yujuan Song
• Afua Nyarko
• Justin Hall
• Greg Benison
• Jessica Morgan
• Invitrogen
• HHMI
• URISC
• OSU Biochemistry and Biophysics Dept.