Term
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Definition
Proteins cannot be synthesized using organic chemistry beyond ~50-70 amino acids. Folding and post-translational modifications may also be an issue
Molecular biology uses PCR, cloning, bacteria, etc. to express proteins |
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Term
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Definition
displaces the lac repressor from the lac operator, activates host T7 RNA polymerase and your gene
Not metabolized |
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Term
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Definition
Lac repressor keeps T7 polymerase (on bacterial chromosome) from being expressed
IPTG removes the Lac repressor, E. coli RNA polymerase makes T7 mRNA, which is translated into T7 RNA polymerase
T7 RNA polymerase finds T7 promoter on the plasmid, transcribes your gene to mRNA, which is translated to protein by E. coli ribosomes |
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Term
| Protein Production in vitro (cell free) |
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Definition
Fast
Expensive and low yield |
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Term
| Protein Production Prokaryotic cells |
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Definition
High levels of expression, techniques have been mastered over the past few decades
Can express proteins toxic to other systems
Bacteria is cheap, can produce it by the liter
Disadvantage: inclusion bodies, improper folding |
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Term
| Protein Production Insect cells (baculovirus) |
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Definition
High yield
Limitless protein size
Efficient cleavage of signal peptides, protein processing
Expression of multiple genes
More difficult than bacterial systems, may modify protein incorrectly or not secrete it |
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Term
| Protein Production Mammalian cells |
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Definition
Get exact protein with all the proper modifications
Slow, expensive, low yield |
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Term
| Protein Production (Best and Worst) |
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Definition
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Term
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Definition
Highly efficient, take up foreign DNA, grow well, easy to regulate, lack proteases, lack recombination
Strains:
BL21 – contain T7 RNA polymerase gene
pLysS – contain plasmid pLysS that expresses T7 lysozyme, which inhibits basal levels of expression (for toxic compounds)
Tuner – Lac permease mutation allows uniform entry of IPTG into cells, allowing you to control induction levels (some proteins require lower concentrations for solubility and activity)
Rosetta – contain pRARE plasmid which encodes tRNAs found mostly in mammals
Origami – thioredoxin reductase, glutathione reductase mutations enhance disulfide bond formation
Mach1 – fast cell growth |
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Term
| Protein Production (General) |
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Definition
Select for bacteria that contain your gene/plasmid of interest with ampicillin
Grow mls to liters. Final amount may vary, but between 1 mg to 500 mg per liter of final pure protein
Grow to an OD600 of ~0.6 (spectrophotometer)
Induce via IPTG
(isopropyl-beta-D-
thiogalactopyranoside)
Spin down cells, lyse |
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Term
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Definition
Pellet cells in centrifuge, discard media
Lyse in correct buffer with protease inhibitors to produce a ‘lysate’ or ‘homogenate’
Mortar and pestle, blender
Freeze/thawing (3X)
Osmotic shock
Chemicals (detergents, organic solvents)
Enzymes
Bacteria – lysozyme
Plants – pectinase, cellulase
Animal – trypsin, hyalurondidase
Sonication
Mechanical devices (tissue homogenizer)
Pressure |
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Term
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Definition
Preparative
Mass production (insulin, enzymes)
Analytical
Identification
Quantification
Post-translational modification
Protein interactions
Structural studies |
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Term
| Early Techniques (Protein Purification) |
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Definition
Centrifugation
Ammonium sulfate cut
Purify protein from cells or organs
Protein purification from chicken gizzards – obtain hundreds of gizzards, add them to a blender, perform ammonium sulfate cuts on resulting lysate
Advantages
Isolate new proteins
No costly or difficult cloning/PCR
Protein is from its natural source (modifications)
Disadvantages
Difficult to obtain large amounts
Difficult to obtain high purity
Protein can aggregate with other proteins
No nickel/GST column selection
Difficult to study human proteins |
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Term
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Definition
Controlled precipitation
Before the days of tags
Perform a gradient – 10%, 20%, 30%, 40%, 50%...
Check each fraction for your protein
[image] |
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Term
| Current Methodology (Protein Purification) |
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Definition
Recombinant DNA technology (cloning, PCR, bacterial transformation, etc)
Advantages
Can obtain exact protein in large amounts
Can engineer mutants to study protein structure/function or enzyme kinetics
Cost-effective
Disadvantages
Must know DNA/protein sequence
Cloning/purification can be tricky
Proper folding?
No post-translational modification |
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Term
| In Vitro Protein Expression |
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Definition
Cell free: mRNA protein directly in tube via ribosomes (or human cell extract)
Advantages
No transformation, cell culture, expression, proteases
High purity, no column chromatography required
Fast (4 hours)
Newest kits claim to produce enough protein for structural studies (5 mg)
Disadvantages
Must have the cDNA or mRNA
of your protein
Current production levels are
on the order of micrograms
Limited protein size (250 kDa)
Kit is expensive ($500) |
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Term
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Definition
Tags
Nickel
GST
MBP
Anion/Cation exchange
Size exclusion
Hydrophobic
Desalting |
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Term
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Definition
| Hsp90b is a molecular chaperone regulating the function of client proteins, including signaling proteins. Hsp90b is isolated from nuclear extracts of HeLa cells through ammonium sulfate fractionation, ion-exchage column chromatographic steps utilizing Q-Sepharose, S-Sepharose, hydroxyapatite, and phosphocellulose, followed by ultracentrifugal separation on a glycerol gradient (20 to 40%). Hsp90b is highly pure and is in a native homodimeric conformation. |
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Term
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Definition
[image]
Best: Dialyze into buffer + dithiothreitol (DTT), store 100 ml aliquots in freezer so only small fractions are removed from the cold at a time |
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Term
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Definition
Purified, highly-concentrated protein in known buffer which can be used in a multitude of laboratory experiments
Molecular weight, other properties
Crystallization/structure
Search for DNA/protein binding partners
Activation/regulation
Enzyme assays
Mutation analysis
Injection |
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Term
| How to remember e to the 15th decimal place. |
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Definition
Jefferson:
7th President
in 1828 x2
45 90 45
2.718281828459045 |
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Term
| Why are GC and HPLC not the best choices for protein purification? |
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Definition
| Too Harsh, will denature proteins |
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