Protein Methods – CHEM 641, 9/17/07 Plasmid isolated 1 Bacterium Bacterial chromosome Plasmid 2 DNA isolated DNA Gene of interest 3 Gene inserted into plasmid Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Copies of geneCopies of protein Clones of cellGene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Cell multiplies with gene of interest Proteins to study in Biochemistry Cell containing gene of interest

Overview of Prokaryotic Expression  Strong promoter – P lac  Ribosome binding – Shine-Dalgarno sequence ~ 7 b.p. before start codon: AUG  Multicloning site to put your gene in with correct frame and direction.

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

Gel Filtration (or size exclusion) Chromatography

Ion Exchange Chromatography

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

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

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

2D PAGE IEF followed by SDS PAGE

Homogeneity / Heterogeneity A.Post translational modification – examples: phosphorylation, glycosylation, myristoylation B.Chemical modifications – cysteine oxidation, Asn/Gln hydrolysis C.Aggregation, unfolding D.Order / disorder E.Alternate Conformations – example hemoglobin bound vs. unbound with oxygen

Next class Protein Structure Determination: X-ray Crystallography, NMR Spectroscopy and Homology Modeling Reading: pgs Lehninger 41/Protein-Structure-and-Function.pdf Class slides: go to CHEM 641 links page