goals for monday 2/18 1.induce, harvest, and lyse bl21de3 cells expressing acetate kinase or acetate...

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

Molybdenum??

http://www.dl.ac.uk/SRS/PX/bsl/scycle.html

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