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Protein Methods – CHEM 641, 9/17/07
1
Bacterium
Plasmid
isolated
2
DNA
isolated
3 Gene
Bacterial
chromosome
Cell containing gene
of interest
inserted
into plasmid
Plasmid
Gene of
interest
Recombinant DNA
(plasmid)
4
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Copies of protein
Clones of cell
Gene used to alter bacteria
for cleaning up toxic waste
Proteins
to study
in Biochemistry
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
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.
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
Gel Filtration (or size exclusion) Chromatography
Ion Exchange Chromatography
Protein’s isoelectric point
Blue – pos.
Red – neg.
Yellow - polar
http://binfo.ym.edu.tw/bioflash/emboss/iep/iep.htm
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
Don’t ever be too sure that its pure enough!
Plasma Platelet Activating Factor Acetylhydrolase gels from Bahnson lab
2D PAGE
IEF followed by SDS PAGE
Homogeneity / Heterogeneity
A. Post translational modification – examples:
phosphorylation, glycosylation, myristoylation
B. Chemical modifications – cysteine oxidation, Asn/Gln
hydrolysis
C. Aggregation, unfolding
D. Order / disorder
E. Alternate Conformations – example hemoglobin
bound vs. unbound with oxygen
Next class
• Protein Structure Determination: X-ray
Crystallography, NMR Spectroscopy and
Homology Modeling
• Reading: pgs 136-139 Lehninger
• http://www.udel.edu/chem/bahnson/Chem6
41/Protein-Structure-and-Function.pdf
• Class slides: go to CHEM 641 links page