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Class overheads for Protein Homogeneity, CHEM 645
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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
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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
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Gel Filtration (or size exclusion) Chromatography
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Ion Exchange Chromatography
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Protein’s isoelectric point
Blue – pos. Red – neg. Yellow - polar
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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
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Don’t ever be too sure that its pure enough!
Plasma Platelet Activating Factor Acetylhydrolase gels from Bahnson lab
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2D PAGE IEF followed by SDS PAGE
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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
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Next class Protein crystallization methods
Field trip to Bahnson lab to grow crystals
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