Goals for Monday 2/18
1. Induce, harvest, and lyse BL21DE3 cells expressing
acetate kinase or acetate kinase variant (manual pg..)
2. Lifetime of protein lab
3. Lecture
4. Measure protein and cytochrome in previous purification
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.
Find conditions for cell growth that enhance a
protein’s 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
photosynthetic conditions
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
Proteins are encoded by genes
5’-GATGCCCCTCGAATAA-3’
3’-CTACGGGGAGCTTATT-5’
DNA
5’-GAUGCCCCAGCAAUAA-3’
mRNA
M—P—Q—Q--STOP
PROTEIN
(PEPTIDE)
Predicted genes or genes of unknown function are typically
called open reading frames (ORF’s)
What do we need to produce a protein?
lamB
A gene
Terminator
Promoter
lamB
Transcriptional unit
Ribosome binding site
lamB
Translational unit
Molecular Biology presents an opportunity for
useful genetic constructs
Antibiotic resistance gene
ori
bla
Origin of
Replication
Plasmid
Terminator
Promoter
lamB
Can fuse gene to other sequences conferring affinity
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
An expression system utilizing lactose and T7 RNA
polymerase is a popular choice in prokaryotes
Genome
ori
bla
Plasmid
lamB
T7 polymerase
dependent promoter
T7 pol
Lactose-inducible
promoter
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.
Additional concerns regarding protein expression
Modifications
Inclusion bodies
Codon usage
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.
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
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
Considering expression systems?
http://www.the-scientist.com/yr1997/sept/profile2_970901.html
http://www.biochem.wisc.edu/biochem660/pdfs/readings/lecture02/Larsen2.pdf
http://www.baculovirus.com/
http://www.biowire.com/bw_jsp/home_top.jsp
http://biobenchelper.hypermart.net/pr/expression.htm
Goals for Tuesday 2/19
1.
2.
3.
4.
5.
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…)
Several hyperthermophilic archaeal species have also been shown
to be dependent on tungsten (W), also Cd important in diatoms
Fe is most abundant, followed by Zn
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:
1. Oxidoreductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligase
Zn is the only element found in all of these classes of enzymes.
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.
Properties of metal ions determine their biological utility
Soft
Hard
Metals favor distinct coordination in proteins
Tetrahedral
Trigonal bipyramidal
L
L
L
M
L
L
L
M
L
L
L
L
L
M
L
L
L
L
M
L
L
Square Planar
M = Metal
L = Ligand
L
Octahedral
L
Unsaturated coordination spheres usually have water as
additional ligands to meet the favored 4 or 6 coordination
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
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
Carbonic Anhydrase catalytic mechanism
Molybdenum??
http://www.dl.ac.uk/SRS/PX/bsl/scycle.html
Tetrapyrroles (heme, chlorophyll) make
proteins “visible” along with certain metals
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
c-type cytochromes have a characteristic
absorbance spectrum
Isobestic point
Purification of cytochrome c2 overview
Cell Fractionation
Protein precipitation
Hydrophobic Interaction chromatography
Gel electrophoresis
Optical spectroscopy
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. purification table, etc.)
Discussion – Implications of results
All lab reports must be type-written (please)
Keeping a purification table
SDS-PAGE examination of purification
1 – Molecular weight markers (see below)
1 2
3
4
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
Periplasmic Fraction
Ammonium Sulfate Fraction
Used 1.0 ml of Ammonium Sulfate
Fraction and phenyl sepharose fraction;
Used a 1:10 dilution of periplasmic
Fraction for these readings
Phenyl Sepharose Fraction