Transcript Chapter 3 (part 2)

Chapter 3 (part 2)
Protein purification and Analysis
Why purify proteins?
• Pure proteins are required to study enzyme
function
• Pure proteins are required for structural
analysis (x-ray crystallography, NMR
spectroscopy)
• Pure proteins are required to obtain amino
acid sequence
Steps in protein purification
• Develop assay
• Choose source of protein
• Prepare tissue extract
– cell disruption
– subcellular fractionation
• Protein fractionation (several steps)
• Determination of purity
Differential Centrifugation
transfer
supernatant
1000 g
tissue
homogenate
10,000 g
Pellet
unbroken cells
nuclei
chloroplast
transfer
supernatant
transfer
supernatant
100,000 g
Pellet
mitochondria
Pellet
microsomal
Fraction
(ER, golgi,
lysosomes,
peroxisomes)
Super.
Cytosol,
Soluble
enzymes
Chromatography
Gel Permeation
Chromatography
Ion-exchange
Chromatography
+++
---
+++
----+ + +++
+ ++ + +
++
+ ++
+ ++ + +
+ ++
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++
low salt buffer
high salt buffer
++
+- ++- -+ +
+ ++
---
+ -+
Cl
Cl+- + +- + Cl- +
++
+ +Cl
+
+
Cl+- + +Cl+
+ ++
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++
++
+ ++ + +
+ ++
+++
+++
++
- +- +++ +
+ ++
++
+ ++ + +
+ ++
+++
---
---
---
Affinity Chromatography
Add excess ligand
SDS poly acrylamide electrophoresis (PAGE)
SDS = H3C-(CH2)10-CH2-OSO3-
SDS – denatures protein
coats w/ negative charge
--- -- - - - -- ---
Used to determine protein MW
And purity of protein prep
Isoelectric Focusing
pH 3
Decreasing pH
-
Decreasing pH
pH 9
+
+
2-D Electrophoresis
Decreasing MW
large
Decreasing pH
Decreasing MW
SDS-PAGE
small
+
Decreasing pH
Amino Acid Analysis
1) Acid hydrolyze protein
2) Treat with phenylisothiocyanate (PICT)
N
C
S
+
H 3N
H
O
C
C
S
C HN
O-
R
C
N
C
O
3) Separate derivatized AA’s by HPLC
R
H
Protein Sequencing
(Edman Degradation)
N
S
C
H 3N
O
C
C
NH
R
2)
N
S
H
O
C HN
C
C
O
C
C
X
R
NH
R
Trifluoroacetic acid
H
H
O
C
C
X
R
S
C HN
3)
C
N
C
R
H
+
2HN
H
O
C
C
X
R
O
Can sequence in 30 to 60 AA’s from N-terminus
Repeat
1)
H
Generate Proteolytic
Fragments
Endopeptidases
•Typsin
•Chymotrypsin
cleaves at COOH end of Lys and Arg
cleaves at COOH end of Phe, Tyr, Trp
Chemical Cleavages
•Cyanogen Bromide
cleaves at COOH end of Met
Generate overlapping fragments
Sequence individual fragments and piece together sequence
Peptide mapping exercise
Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp
Trypsin
Met-Ala-Arg
Phe-Ala-Glu-Gln-Asp
Gly-Glu-Tyr-Met-Cys-Lys
Chymotrysin
Met-Ala-Arg- Gly-Glu-Tyr
Met-Cys-Lys –Phe
Ala-Glu-Gln-Asp
CNBr
Met
Ala-Arg-Gly-Glu-Tyr-Met
Cys-Lys-Phe-Ala-Glu-Gln-Asp
Proteomic Analysis
Matrix Assisted Laser Desorption Ionization Time of Flight
(MALDI-TOF)