1. Class overheads for Protein Homogeneity, CHEM 645

2. Overview of Prokaryotic Expression
Strong promoter – Plac
Ribosome binding – Shine-Dalgarno sequence
~ 7 b.p. before start codon: AUG
Multicloning site to put your gene in with correct frame and direction.
Class overheads for Protein Homogeneity, CHEM 645

3. Affinity Chromatography using fusion proteins
Construct a fusion of
affinity tag with your protein
Add a protease cleavage
site (thrombin)
Express fusion protein
Purify by affinity
chromatography
Cleave tag
Examples:
His-tag,
GST fusion,
maltose binding protein fusion

4. Gel Filtration (or size exclusion) Chromatography

5. Ion Exchange Chromatography

6. Protein’s isoelectric point
Blue – pos.
Red – neg.
Yellow - polar

8. SDS PAGE SDS-sodium docecylsulfate Denaturing conditions Boil 100 ºC
DTT, b-mercaptoethanol
Cys-S-S-Cys  Cys-SH
Elution rate  to log MW
MWM crude fusion cleaved protein of interest

9. Don’t ever be too sure that its pure enough!
Plasma Platelet Activating Factor Acetylhydrolase gels from Bahnson lab

10. 2D PAGE
IEF followed by SDS PAGE

11. Homogeneity / Heterogeneity
Post translational modification – examples: phosphorylation, glycosylation, myristoylation
Chemical modifications – cysteine oxidation, Asn/Gln hydrolysis
Aggregation, unfolding
Order / disorder
Alternate Conformations – malate dehydrogenase loop

12. Next class Protein crystallization methods
Field trip to Bahnson lab to grow crystals