Protein purification

in practice

• Quantification of the procedure – How well did it work?

– Did something go wrong? Where?

• Know

how much

fumarase is present at each step – “Yield” – Total enzyme activity ( m mol malate/min) – Need activity of a certain volume of the fraction and the volume of the fraction.

• Know

how pure

fumarase is at each step – “Specific activity” – Enzyme activity per mg of total protein • For each major sample, record: – activity ( D abs/time): specifically how much fumarase is present – protein concentration (Bradford): total indiscriminate protein – volume

Starting material SAMPLE 1 SAMPLE 2 Which has a higher

yield

of fumarase?

Which will show a higher

specific activity

?

Which has a higher

fold purification

?

Major Samples

• Crude extract – How much fumarase did you start with?

– aka “source material”, “pre-column extract”, “applied” • Purified enzyme x 2 – pre- and post-dialysis) – How much did you recover?

Major Samples

• Crude extract • Flow-through – How much fumarase bound to the column?

– How much flowed through the column?

– “Post-column extract” – What

should

the specific activity of the FT be?

• Purified enzyme

“Purification Table”

Need to know mg/mL (Bradford) Crude Column Flow Through Purified (Pre dialysis) Purified (Post-dialysis) Volume Total Protein (mg) Total Activity ( m mol/min) Specific Activity ( m mol/min/mg) Yield (%) Fold Purification

General procedure

• Prepare column – “Flow adaptor” – No air – Wash column briefly with extraction buffer • Prepare sample – Re-centrifuge • Start collecting fractions!

• Apply sample to column • Wash column with extraction buffer • Elute fumarase with several “column volumes” of elution buffer (imidazole) – Collect smaller fractions (~1.5 mL)

Measure A280s of fractions

Flow Through “Wash” Start elution (500 mM Imidazole) Fraction number Wash: remove

all

low-affinity proteins from the column A280 should return ~ baseline Elute: Protein (fumarase) should be collected A280 ≠ protein here

Imidazole elutions

• Contain fumarase (theoretically) • Contain ~500 mM Imidazole – May

inhibit

fumarase activity • Give falsely low reading for yield, specific activity?

• “Dialyze” the protein – Lower the imidazole concentration

Dialysis of biological molecules

• Not really a purification procedure – Removes

salts

& other small molecules – Not designed to remove other

proteins

• Place protein solution in a “bag” made from a semipermeable membrane – Permeable to water and

small

molecules • Place dialysis bag in a container with lower salt buffer – Diffusion/osmosis for system to achieve osmotic balance • Salts/small molecules diffuse out • Water diffuses in

Beginning dialysis After equilibration

Dialysis ~ Dilution

• Start with 3 mL of fumarase with 500 mM Imidazole • Dialyze overnight in 500 mL of dialysis buffer (no Imidazole) • What’s the final concentration of Imidazole in the sample?

Beginning dialysis After equilibration 3 mL 500 mM Imidazole 500 mL ?

Fractions you’ll have

• Crude (stored at 4°) (save a few hundred m L) • Crude stored frozen • Flow through (pool from fraction collector) • Eluted fumarase – Assays to determine which fractions contain fumarase – Pool fumarase – Save a few hundred m L – Dialyze the rest • Dialyzed fumarase