goals for monday 2/18 1.induce, harvest, and lyse bl21de3 cells expressing acetate kinase or acetate...
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Goals for Monday 2/18
1. Induce, harvest, and lyse BL21DE3 cells expressingacetate kinase or acetate kinase variant (manual pg..)
2. Lifetime of protein lab3. Lecture4. Measure protein and cytochrome in previous purification
We can “control” protein expression
With the notable exception of proteins such asthose that compose the ribosome, many proteinsare found only in low abundance (particularly proteinsinvolved in regulatory processes)
Thus, we need to find ways to grow cells that allow ampleexpression 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.sphaeroidesfor 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 synthesizedunder photosynthetic growth conditions; obviously if you want to purify this protein you need to grow cells underphotosynthetic conditions
Molecular Biology allows us to manipulate genes
Understanding the basic mechanisms of gene expressionhas allowed investigators to exploit various systems for protein expression
Prokaryotic expression systems
Eukaryotic expression systemsYeastMammalian
Viral expression systemsBaculovirus and Insects
Proteins are encoded by genes
M—P—Q—Q--STOP PROTEIN(PEPTIDE)
5’-GATGCCCCTCGAATAA-3’3’-CTACGGGGAGCTTATT-5’
DNA
5’-GAUGCCCCAGCAAUAA-3’ mRNA
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
lamB
Promoter
Transcriptional unit
Terminator
lamB
Ribosome binding site
Translational unit
Molecular Biology presents an opportunity for useful genetic constructs
lamB
Promoter Terminator
bla
Plasmid
oriAntibiotic resistance gene Origin ofReplication
Can fuse gene to other sequences conferring affinity
Choice of promoter allows control over transcription levels
Intrinsic promoters can be sufficient for overexpressionin multi-copy plasmids
Constitutive promoters with high activity (ie. promoters forribosomal genes) can be useful for producing non-toxicproteins
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
lamB
blaori
Plasmid
T7 polymerasedependent promoter
T7 polLactose-inducible promoter
Genome
Inclusion bodies provide a rapid purification step
Proteins existas aggregates in inclusionbodies thus special precautions must be takenduring purification. Typically,inclusion bodies can be readilyisolated via cell fractionation.following isolation the proteinsmust be denatured and renaturedto 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 ofheterologous protein expression may contain“rare” codons that are not normally utilized bycells such as E. coli. Thus, this could limit protein production. Codon usage has been usedfor determination of highly expressed proteins.
Molecular Biology allows us to manipulate genes
Understanding the basic mechanisms of gene expressionhas allowed investigators to exploit various systems for protein expression
Prokaryotic expression systems
Eukaryotic expression systemsYeastMammalian
Viral expression systemsBaculovirus and Insects
Non-prokaryotic expression systems have emerged due toincreasing 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
http://www.the-scientist.com/yr1997/sept/profile2_970901.html
Considering expression systems?
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. Lecture2. Load and run SDS-PAGE gel for cytochrome purification3. Prepare crude extracts (centrifugation)4. Purify His-tagged proteins5. 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 forchemistry, and often aid in structural stabilization.
Protein enzymes are divided into six classes by the Enzyme Commision:1. Oxidoreductase2. Transferase3. Hydrolase4. Lyase5. Isomerase6. 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 chargeionic radii, and ionization potential
Typically, metals are classified as “hard” or “soft” incorrelation with their ionic radii, electrostatics, andpolarization
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
M
LL
L L
M
L L
LLM
L
LL
LL
LM
L
L L
L
L
Tetrahedral
Square Planar
Trigonal bipyramidal
Octahedral
M = MetalL = Ligand
Unsaturated coordination spheres usually have water as additional ligands to meet the favored 4 or 6 coordination
Protein sequence analyses have revealed certain metalbinding 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
CatalyticL1-few aa-L2-several aa-L3
StructuralL1-3-L2-3-L3-8-L4
L = Ligand
Biological roles of transition metals
Coordination Structure (protein and protein-substrate)Electrophilic catalysis Positive charge attracts electrons, polarize potential reactant, increase reactivityGeneral Acid – Base catalysisRedox reactionsMetalloorganic chemistry Free radicals
(not just limited to proteins*)
Carbonic Anhydrase catalytic mechanism
Tetrapyrroles (heme, chlorophyll) makeproteins “visible” along with certain metals
Spectroscopy is a study of the interaction of electromagnetic radiation with matter
A = cl
Absorbance = extinction coefficient x concentration x path length
Beer-Lambert Law
The amount of light absorbed is proportional to the number ofmolecules of the chromophore, through which the light passes
Units: None = M-1 cm-1 M cm
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 experimentsare important (not simply from a course workperspective)
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)
Keeping a purification table
SDS-PAGE examination of purification
1 2 3 41 – Molecular weight markers (see below)
2 – Periplasm fraction
3 – Ammonium sulfate fraction
4 – Phenyl Sepharose fraction
MW marker sizes:
97.4 kDa66.2 kDa45 kDa31 kDa21.5 kDa14.4kDa
Explain these results in yourlab report
Periplasmic Fraction Ammonium Sulfate Fraction
Phenyl Sepharose Fraction
Used 1.0 ml of Ammonium SulfateFraction and phenyl sepharose fraction;Used a 1:10 dilution of periplasmic Fraction for these readings