Protein Methods CHEM 641 91707 1 Bacterium Plasmid

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

Overview of Prokaryotic Expression ØStrong promoter – Plac ØRibosome binding – Shine-Dalgarno sequence ~ 7 b. p. before start codon: AUG ØMulticloning site to put your gene in with correct frame and direction.

Affinity Chromatography using fusion proteins q. Construct a fusion of affinity tag with your protein q. Add a protease cleavage site (thrombin) q. Express fusion protein q. Purify by affinity chromatography q. 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 http: //binfo. ym. edu. tw/bioflash/emboss/iep. htm

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

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

2 D 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 136 -139 Lehninger • http: //www. udel. edu/chem/bahnson/Chem 6 41/Protein-Structure-and-Function. pdf • Class slides: go to CHEM 641 links page