Goals for Monday 2/18

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Goals for Monday 2/18

• Induce, harvest, and lyse BL21DE3 cells
  expressing
• acetate kinase or acetate kinase variant (manual
  pg..)
• Lifetime of protein lab
• Lecture
• Measure protein and cytochrome in previous
  purification

2
We can control protein expression
With the notable exception of proteins such
as those that compose the ribosome, many
proteins are found only in low abundance
(particularly proteins involved in regulatory
processes) Thus, we need to find ways to grow
cells that allow ample expression of proteins
that would be interesting for biochemical
characterization.
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Find conditions for cell growth that enhance a
proteins expression
For example, cytochrome c2 is utilized by
R.sphaeroides for both respiratory and
photosynthetic growth a slight increase in
levels of this protein is observed under
photosynthetic growth conditions. However,
Light-Harvesting complexes are only
synthesized under photosynthetic growth
conditions obviously if you want to purify this
protein you need to grow cells under photosyntheti
c conditions
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Molecular Biology allows us to manipulate genes

• Understanding the basic mechanisms of gene
  expression
• has allowed investigators to exploit various
  systems for
• protein expression
• Prokaryotic expression systems
• Eukaryotic expression systems
• Yeast
• Mammalian
• Viral expression systems
• Baculovirus and Insects

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Proteins are encoded by genes
Predicted genes or genes of unknown function are
typically called open reading
frames (ORFs)
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What do we need to produce a protein?
lamB
A gene
Ribosome binding site
lamB
Translational unit
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Molecular Biology presents an opportunity for
useful genetic constructs
Antibiotic resistance gene
Origin of Replication
ori
bla
Plasmid
Can fuse gene to other sequences conferring
affinity
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Choice of promoter allows control over
transcription levels

• Intrinsic promoters can be sufficient for
  overexpression
• in multi-copy plasmids
• Constitutive promoters with high activity (ie.
  promoters for
• ribosomal genes) can be useful for producing
  non-toxic
• proteins
• Inducible promoters allow control of expression,
  one can
• titrate the promoter activity using
  exogenous agents

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An expression system utilizing lactose and T7
RNA polymerase is a popular choice in
prokaryotes
Genome
Plasmid
T7 polymerase dependent promoter
T7 pol
Lactose-inducible promoter
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Inclusion bodies provide a rapid purification step
Proteins exist as aggregates in inclusion bodies
thus special precautions must be taken during
purification. Typically, inclusion bodies can be
readily isolated via cell fractionation. following
isolation the proteins must be denatured and
renatured to retrieve active protein.
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Additional concerns regarding protein expression
Modifications Inclusion bodies Codon usage
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Cells exhibit nonrandom usage of codons
This provides a mechanism for regulation however,
genes cloned for purposes of heterologous
protein expression may contain rare codons that
are not normally utilized by cells such as E.
coli. Thus, this could limit protein
production. Codon usage has been used for
determination of highly expressed proteins.
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Molecular Biology allows us to manipulate genes

• Understanding the basic mechanisms of gene
  expression
• has allowed investigators to exploit various
  systems for
• protein expression
• Prokaryotic expression systems
• Eukaryotic expression systems
• Yeast
• Mammalian
• Viral expression systems
• Baculovirus and Insects

15
Non-prokaryotic expression systems have emerged
due to increasing simplicity and the need for
proper modifications.
Although you can express a eukaryotic cDNA in a
prokaryote is the protein you purify, what the
eukaryotic cell uses?
Invitrogen www.invitrogen.com Gateway
vectors Novagen www.novagen.com
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Considering expression systems?
http//www.the-scientist.com/yr1997/sept/profile2_
970901.html
http//www.biochem.wisc.edu/biochem660/pdfs/readin
gs/lecture02/Larsen2.pdf
http//www.baculovirus.com/
http//www.biowire.com/bw_jsp/home_top.jsp
http//biobenchelper.hypermart.net/pr/expression.h
tm
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Goals for Tuesday 2/19

• Lecture
• Load and run SDS-PAGE gel for cytochrome
  purification
• Prepare crude extracts (centrifugation)
• Purify His-tagged proteins
• Generate standard curve for acetyl-phosphate
  determinations
• (pg)

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Several hyperthermophilic archaeal species have
also been shown to be dependent on tungsten (W),
also Cd important in diatoms
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Fe is most abundant, followed by Zn
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Metals in Biology

• All ribozymes are metalloenzymes, divalent
  cations are required for
• chemistry, and often aid in structural
  stabilization.
• Protein enzymes are divided into six classes by
  the Enzyme Commision
• Oxidoreductase
• Transferase
• Hydrolase
• Lyase
• Isomerase
• Ligase
• Zn is the only element found in all of these
  classes of enzymes.

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Proteins bind metals based on size, charge,
and chemical nature
Each metal has unique properties regarding ionic
charge ionic radii, and ionization potential
Typically, metals are classified as hard or
soft in correlation with their ionic radii,
electrostatics, and polarization
Hard metals prefer hard ligands, soft prefer
soft, Borderline metals can go either way.
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Properties of metal ions determine their
biological utility
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Soft
Hard
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Metals favor distinct coordination in proteins
Tetrahedral
Trigonal bipyramidal
M Metal L Ligand
Octahedral
Square Planar
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Unsaturated coordination spheres usually have
water as additional ligands to meet the favored
4 or 6 coordination
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Protein sequence analyses have revealed certain
metal binding motifs
Structural Zn are generally bound by 4
cysteines Catalytic Zn bound by three residues
(H, D, E, or C) and one water
Coordination in primary sequence of alcohol
dehydrogenase Catalytic L1-few aa-L2-several
aa-L3 Structural L1-3-L2-3-L3-8-L4
L Ligand
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Biological roles of transition metals
(not just limited to proteins)

• Coordination
• Structure (protein and protein-substrate)
• Electrophilic catalysis
• Positive charge attracts electrons, polarize
• potential reactant, increase reactivity
• General Acid Base catalysis
• Redox reactions
• Metalloorganic chemistry
• Free radicals

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Carbonic Anhydrase catalytic mechanism
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Molybdenum??
http//www.dl.ac.uk/SRS/PX/bsl/scycle.html
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Tetrapyrroles (heme, chlorophyll) make proteins
visible along with certain metals
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Spectroscopy is a study of the interaction of
electromagnetic radiation with matter
A ecl
Absorbance extinction coefficient x
concentration x path length
Units None M-1 cm-1
M cm
Beer-Lambert Law
The amount of light absorbed is proportional to
the number of molecules of the chromophore,
through which the light passes
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c-type cytochromes have a characteristic
absorbance spectrum
Isobestic point
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Purification of cytochrome c2 overview

• Cell Fractionation
• Protein precipitation
• Hydrophobic Interaction chromatography
• Gel electrophoresis
• Optical spectroscopy

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Lab reports
Introduction Rationale for why these
experiments are important (not simply from a
course work perspective) Materials Methods
Concise, but detailed description of how
experiments were performed Results Summary of
data (Simply report data, ie. purifica- tion
table, etc.) Discussion Implications of
results All lab reports must be type-written
(please)
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Keeping a purification table
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SDS-PAGE examination of purification
1 Molecular weight markers (see below) 2
Periplasm fraction 3 Ammonium sulfate
fraction 4 Phenyl Sepharose fraction MW
marker sizes 97.4 kDa 66.2 kDa 45 kDa 31
kDa 21.5 kDa 14.4kDa
Explain these results in your lab report
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Periplasmic Fraction
Ammonium Sulfate Fraction
Used 1.0 ml of Ammonium Sulfate Fraction and
phenyl sepharose fraction Used a 110 dilution
of periplasmic Fraction for these readings
Phenyl Sepharose Fraction