Experiment 3: Purification of Fumarase

1. Experiment 3: Purification of Fumarase • Three week experiment • Wraps up with formal lab report • Abstract/Intro/Results/Discussion would be difficult to write super early • Keep up with M & M • Think about/work on intro? • DAQs after week 1 • Week 3 “DAQs” in lab report

2. Protein Explorer exercise • Assignment tomorrow • Due date will be pushed back a couple of days (originally Friday, March 16th) • Grading • Exams tomorrow • Paper summaries • Not even close

3. Why purify a protein? • Determine amino acid sequence (1° structure) • Determine 3-D structure (2°, 3°, 4°) • Function of protein away from cellular partners (in vitro) • Interactions with other molecules • Enzyme catalysis • Inhibitors (drugs?)

4. Four ‘goals’ this week • Lyse yeast • Determine how much fumarase you start with • Set up Nickel column • Set up/become familiar with pump/fraction collector • Anion exchange ‘trial’

5. 1st step of protein purification Lysis Centrifugation Get rid of insoluble cellular components -cell wall/membrane -organelles Soluble proteins

6. How are cells/tissues lysed? • Enzymatic/chemical • Degrade cell membrane • Detergents/lytic enzymes • Mechanical • eg. Sonication (high energy sound waves)

7. Yeast are very tough: cell wall • Mechanical lysis: Bead mill • Yeast + Glass beads + “Blender” • Lysis buffer • Bit of detergent to weaken the cell (& stabilize protein) • Phosphate at ~ neutral pH, some salt • Protease inhibitors

8. Purify/separate fumarase • Physical/chemical characteristics: make it ‘unique’ among cellular proteins • Here: we’ve altered the characteristics of fumarase • Genetic manipulation • Add a ligand binding site…affinity purification

9. Purify/separate fumarase Genomic (native) gene for fumarase (DNA) Codes for fumarase (protein) Could be purified based on -size -pI/charge -etc.

10. Purify/separate fumarase Manipulated gene +~18 additional nucleotides Codes for fumarase + ~6 additional amino acids at the C terminus H-H-H-H-H-H We’ve added a gene to this strain of yeast! Unique feature among proteins

11. Problem of protein stability • “Proteolysis” • Lysis of peptide bonds • Typically catalyzed: proteases • KEEP PROTEIN COLD • Slow down proteolytic enzymes • Chemical protease inhibitors • Block enzyme action

12. Problem of protein stability • Structural stability • Native-like buffers • Prevent drastic changes in pH, osmolarity • Detergents can stabilize protein structure • *Mild* eg. Igepal, Tween-20, etc. • Many detergents destabilize protein structure, eg. SDS • Temperature • Room temp too warm? • Centrifugation • Bead milling • Storage? Generate lots of heat

13. *Fumarase: relatively high affinity for stationary phase *Other proteins: mobile phase *Weak fumarase’s affinity by competition INSOLUBLE “Bead” H2O H2O COVALENT http://tinyurl.com/ysn9ya Company’s handbook (Qiagen)

14. Fraction collector • Pump • Gradient maker

15. Anion exchange trial • Start with three proteins • Affinity for stationary vs. mobile phases (partitioning) • Change stationary phase/change partitioning • Watch volume used for gradient

16. A: 25 mM Tris B: 25 mM Tris, 500 mM NaCl “Gradient maker” Equal volumes To column Start: 100% A Column sees 0 mM NaCl B A Volumes decreased but still equal Column sees 250 mM NaCl To column Halfway through: 50% A B 50%A 50%B Column sees ~500 mM NaCl To column Close to the end: almost 100% B B >100%B