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Class overheads for Protein Homogeneity, CHEM 645

Class overheads for Protein Homogeneity, CHEM 645. http://molvis.sdsc.edu/protexpl/xtlcon.htm. Overview of Prokaryotic Expression. Strong promoter – P lac Ribosome binding – Shine-Dalgarno sequence ~ 7 b.p. before start codon: AUG

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Class overheads for Protein Homogeneity, CHEM 645

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  1. Class overheads for Protein Homogeneity, CHEM 645 http://molvis.sdsc.edu/protexpl/xtlcon.htm

  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 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 – malate dehydrogenase loop

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

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