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Protein Methods – CHEM 641, 9/17/07

Explore methods of gene insertion into bacteria for protein production, including affinity chromatography and chromatography techniques. Learn about protein structure determination and post-translational modifications.

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Protein Methods – CHEM 641, 9/17/07

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  1. 1 Bacterium Plasmidisolated 2 DNA isolated 3 Gene inserted into plasmid Bacterialchromosome Plasmid Gene ofinterest Recombinant DNA(plasmid) DNA 4 Plasmid put intobacterial cell Recombinantbacterium 5 Cell multiplies withgene of interest Proteins to study in Biochemistry Copies of gene Copies of protein Gene for pestresistanceinserted intoplants Clones of cell Protein used to make snow format highertemperature Gene used to alter bacteriafor cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy Protein Methods – CHEM 641, 9/17/07 Cell containing geneof interest

  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.

  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 http://binfo.ym.edu.tw/bioflash/emboss/iep/iep.htm

  7. 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

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

  9. 2D PAGE IEF followed by SDS PAGE

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

  11. Next class • Protein Structure Determination: X-ray Crystallography, NMR Spectroscopy and Homology Modeling • Reading: pgs 136-139 Lehninger • http://www.udel.edu/chem/bahnson/Chem641/Protein-Structure-and-Function.pdf • Class slides: go to CHEM 641 links page

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