E XPLORING P ROTEINS AND P ROTEOMES. G ENOMES AND P ROTEOMES.

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Presentation transcript:

E XPLORING P ROTEINS AND P ROTEOMES

G ENOMES AND P ROTEOMES

O BTAINING THE PROTEIN : CELL LYSIS AND S EPARATION

P ROTEIN P URIFICATION T ECHNIQUES Salting out Dialysis Gel Filtration Chromatography Ion-exchange Chromatography Affinity Chromatography HPLC Gel Electrophoresis Isoelectric Focusing 2D Gel Electrophoresis Ultracentrifugation

S ALTING O UT

D IALYSIS

G EL F ILTRATION C HROMATOGRAPHY

I ON - EXCHANGE C HROMATOGRAPHY

A FFINITY C HROMATOGRAPHY

ELISA

HPLC

G EL E LECTROPHORESIS

I SOELECTRIC F OCUSING

2D G EL E LECTROPHORESIS

U LTRACENTRIFUGATION

S TRUCTURE D ETERMINATION

A MINO A CID S EQUENCING : E DMAN D EGRADATION

B ROAD C LASSIFICATION OF P ROTEASE Serine Proteases A serine residue acts as a nucleophile in the active site, facilitating the reaction Operates via the catalytic triad His, Ser- Asp Cystein Proteases Cys as the nucleophile and activated by a nearby basic aa Papain is an example Aspartate Proteases Two Asp residues cleaves the peptide bond by activating a water molecule Metalloprotease A metallic Zn or Co is involved in the catalytic mechanism

E XAMPLES OF P ROTEASES Trypsin – carboxyl side of Lys and Arg Chymotrypsin – carboxyl side of bulky hydrophobic aa Elastase – carboxyl side of small hydrophobic aa Clostripain – carboxyl side of Arg Thrombin – serine protease with specific cleavage site: Leu-Val-Pro-Arg—Gly-Ser Carboxypeptidase A – amino side of C terminal residues of aromatic or aliphatic aa An exopeptidase

M ASS S PECTROMETRY

MALDI-TOF MS

E LECTROSPRAY I ONIZATION

X- RAY C RYSTALLOGRAPHY

X- RAY D IFFRACTION P ATTERN OF M EVALONATE K INASE

NMR S PECTROSCOPY