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QBM exam Prep Test| Questions Solved with Verified Correct Answers | Latest Update
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QBM exam Prep Test| Questions Solvedwith Verified Correct Answers | Latest
Update 2024-2025
What advantages does the phage expression system offer over the Ptac system? - ANSWER Ptac
system can be leaky, has low levels of protein of interest, even in the "off" state, can result in
misfoled protein
Phage system has an extra step, prevents background expression, T7 lysozome prevents
this even further
Describe the phage expression system once IPTG is added to the media: - ANSWER E.coli
takes up IPTG, binds to the lac repressor, removes the repressor from the operator.
,E.coli RNA polymerase synthesizes T7 RNA polymerase which targets the T7 promotor
on plasmid & transcribes mRNA from gene of interest.
E.coli ribosomes transcribe this mRNA to protein.
Some labs use pGEX vector (w/ its Ptac promotor) in E.coli BL21 cells. What's the flaw in this? -
ANSWER Upon induction, IPTG removes the repressor from the BL21 genomic DNA, allowing
for T7 transcription/translation.
The Ptac vector doesn't require T7, it's repressor is also removed by IPTG & gene of interest
is transcribed by E.coli polymerases.
Therefore, using BL21 & T7 polymerases w/ Ptac promotor isn't required & may reduce
expression efficiency as the bacterial cell must also devote resources into synthesizing T7,
which is wasteful.
Protease Inhibitors: - ANSWER - Added to buffer in which lysed proteins are placed to prevent
degradation
- PMSF
- EDTA
- EGTA
Lysing Bacterial Cells: Best--> Worst - ANSWER 1. French Press: very high pressure (40,000 psi),
result can be very viscous
2. Sonicator: for short bursts & on ice to prevent overheating
3. Chemical lysis: can cause osmotic shock (good for lysing), can interfere w/ downstream
techniques (Bradford)
4. Lysozyme
5. Freeze/thawing: 3 cycles
,6. Mortar & Pestle (Blender): older technique, doesn't work for bacterial cells, used
for eukaryotic
7. Enzymes: for small volumes of cells
Bacteria - lysozyme
Plants - pectinase, cellulase
Animal - trypsin, hyalurondidase
8. Tissue homogenizer: shearing/ spinning cells at high rpm; limited to eukaryotic cells
Protein Purification: - ANSWER - Preparative: Mass production (insulin, enzymes)
- Analytical:
Identification
Quantification
Post-translational
modification Protein
interactions Structural studies
Early, modern, and
Why do researchers use a purification table? - ANSWER To calculate & track each step
of protein purification & calculate final yield
Ammonium Sulfate Cut: - ANSWER - Controlled precipitation where proteins are
gently removed from buffer & stabilized by (+) on ammonium & negative on sulfate
- Purify protein from cells or organs
- Protein purification from organs - i.e. obtain hundreds of chicken gizzards, blend
them, perform ammonium sulfate cuts on resulting lysate/homogenate
, Advantages & Disadvantages of Ammonium Sulfate Cut: - ANSWER - Advantages:
- Isolate new proteins
- not costly or difficult cloning/PCR
- protein is from its natural source (modifications)
- structure forming salt, stabilizes protein even w/o H2O
- 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
Ammonium Sulfate Cut (Fractionation) - ANSWER - Controlled precipitation using structure-
forming salts to increase the level of intermolecular structures in water, the salts increase
- Hydrophobic interactions
- Used to clarify crude extracts
- Stabilizes proteins
- Gentle method for purification
- Perform a gradient - 10%, 20%, 30%, 40%, 50%, check fractions
- If you add TOO MUCH salt, protein will come out as a precipitant
Recombinant DNA technology: - ANSWER Advantages: - Can obtain exact protein in large
amounts
- Can engineer mutants to study protein structure/function or enzyme kinetics
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