5 | Preparative Protein Biochemistry Flashcards
(96 cards)
1
Q
What is the only truly accurate method for determining protein concentration?
A
Acid hydrolysis followed by amino acid assay
2
Q
What method make use of UV absorption in protein conc. determination?
A
Nanodrop
3
Q
Lowry Assay can detect down to, and at what absorbance?
A
10 μm cm−3, 660 nm
4
Q
In Lowry assay what is happening in the Folin–Ciocalteau reaction?
A
copper-catalysed oxidation of aromatic amino acids
5
Q
What colour does the Lowry assay product give?
A
Blue
6
Q
BCA assay can detect down to, and at what absorbance?
A
0.5 μm cm−3, 562 nm
7
Q
What is the difference between Lowry and BCA? (2)
A
In BCA besides the folin C reaction we also have biuret reaction
8
Q
What colour does the BCA assay product give?
A
purple
9
Q
Bradford method can detect down to, and at what absorbance?
A
20 μm cm−3, 595 nm
10
Q
What is the practical advantages of using the Bradford method? (3)
A
Its reliable because its simple in nature giving stable outcomes and rapid development
11
Q
What two problems are apparent with Bradford method?
A
- Amount of dye binding appears to vary with arginine and lysine content
- many proteins will not dissolve properly in the acidic reaction medium.
12
Q
Name a few affinity tags?
A
GST, His-tag, MBP
13
Q
What compound is linked to agarose beads when we are using a GST-fused protein construct?
A
glutathione
14
Q
What compound is added in excess to elute GST-fusion protein after wash buffer step?
A
Reduced glutathione
15
Q
How does one remove the non-protein from the matrix?
A
With wash buffer
16
Q
At what terminal to the target gene is GST typically fused via?
A
N
17
Q
Is fusion constructs with GST more soluble than with MBP, wrt. the native form?
A
No, vice versa
18
Q
At what terminal to the target gene should MBP be fused via? to aid in the folding of the target protein
A
N
19
Q
When is it preferable to use MBP over His-tag and/or GST?
A
When we’re working with “difficult” proteins that fail to be expressed.
20
Q
What does the imidazole ring in the essential amino acid histidine provide and enable, wrt. the His-tag?
A
It enables co-ordinated metal ion binding in a pH-dependent manner giving a stable structure.
21
Q
To what does the His-tag confer binding to?
A
Immobilized bivalent nickel or cobalt ions
22
Q
How can His-tagged proteins be eluted with?
A
Free imidazole or at acidic pH
23
Q
How can one overcome the rel. poor solubility of TEV protease?
A
By expressing TEV protease in a bacterial culture as an MBP fusion construct with a TEV protease recognition site
24
Q
What chromatographic technique under purification pipeline considerations make use of His-tagged TEV protease constructs?
A
Immobilized metal ion chromatography
25
As discussed above, it is possible to create fusion proteins by engineering the DNA to contain the required sequence. However, it may be desirable to introduce ___ ___ that cannot be achieved through molecular cloning or reliably achieved through chemical or enzymatic modification.
posttranslational
modifications
26
For cloned genes that are being expressed in microbial or eukaryotic cells, there are several advantages (3) in manipulating the gene to ensure that the protein product is secreted from the cell:
1. Whether the protein is secreted into a growth medium isa more feasible than it to be released by cell rupture making it possible for contaminating intracellular proteins to be present.
2. We want to mitigate degradation by intracellular proteases.
3. Some cloned proteins are toxic to the cell which may induce cell death.
27
What does the lac operon drive ?
Synthesise IPTG that relieve repression of the lac repressor --> drive transcription and translation of desired gene in E. coli
28
What’s the difference between Gram-positive/negative bacteria?
1. Peptidoglycan cell wall, Cytoplasmic membrane
2.LPS, Outer membrane, BLP, Peptidoglycan cell wall, periplasm, cytoplasmic membrane
29
Having a plasmid containing the gene of interest that has been cloned into an appropriate expression vector, how can it be transformed into the bacterial cells?
Heat shock or electroporation
30
How can we improve cell viability?
Chosing gentle initial culturing conditions, using an enriched culture media.
31
Why is it so that there’s a lack of success for successful over-expression of recombinant genes in bacteria? (2)
1. sequence of amino acids containing significant secondary structure that inhibit correct protein folding
2. the gene of interest may utilise rare codons that exist in the source organism, but do not occur in the bacterial expression organism
32
A successful protein purification is more likely to be achieved if the starting material contains …
Proper choice of promoter system in biotechnologically used plasmids is important to think of in order to mitigate unwanted basal expression, which is common in most strains of E. coli. Thus the choice of bacterial strain and promotor system need to work together
33
How do you avoid cellular stress that may be caused by over-expression of the target gene?
The media used for growing the bacterial culture contains the appropriate antibiotic for selection and the cells are typically grown in a shaker flask until an optical density of OD 600 = 0.5–1.0 is achieved
34
With regard to media used for growing bacterial culture, what component is added to media for selection? And why do we seek a OD600 = 0.5 -1.0?
Antibiotics, In order to avoid cellular stress when we want to over-express a specific target gene.
35
Why is it so that the choice of bacterial strain has a large impact on protein expression?
Because we want to avoid basal level of expression and mitigate cellular stress
36
In a general sense what 3 factors come to play when we speak of commercially available optimized bacterial strains?
Because we want to make sure the plasmid is able to encode even rare codons, but also having strong repressor function to avoid basal expression and lastly be able to be highly competent (i.e. easy to transform)
37
Why are bacterial cultures commonly grown in shaker flasks?
During growth we want to increase yield that’s why there’s an advantage in considering oxygenation of the culture media
38
Before one can grow large cultures, the transformed bacteria are grown in an enriched medium called?
SOC – which allows viable bacteria to grow before plating out.
39
Why is there a deliberate absence of activating sugars in LB ? and what does IPTG have to do with this?
In order to prevent basal expression of the plasmid, so that IPTG can be added to cause protein expression
40
Why would we employ auto-induction media?
Because it contains a carefully defined amount of glucose, that is preferentially used by the bacteria until becoming exhausted at the point just before saturation. At this moment, other sugars will induce their operons and cause expression of the target gene without the need to add IPTG.
41
Describe the cell wall of yeast and fungi?
1. Fungi: Cell membrane, chitin, beta-glucans, mannoproteins
2. Yeast: Cell membrane, larger % Chitin, beta-glucans, mannoproteins
42
What is the major advantage (1) and disadvantages (2) of using mammalian cells?
1. increased chances of achieving correct folding and the post-translational modifications that may be required
2. high costs of media and the potentially more complex chromatography protocols required. In addition the cells lack any substantial rigidity thus more easy to disrupt by shear forces.
43
What is the cell wall composed of in plant cells?
Plasma membrane, primary cell wall (cellulose microfibrils, cross-linking glycan, pectin), middle lamella
44
What two buffers are most commonly used?
TRIS & phosphate buffer
45
Give examples of reagents that may be added to the buffer for specific purposes?
Antioxidant, enzyme inhibitors, enzyme substrate and cofactors, phosphatase inhibitors, EDTA
46
Within the cell, proteins are in a fairly ____ environment, but when released into the buffer they are exposed to a more
____environment.
1. reducing
2. oxidative
47
Why would we add an antioxidant to a extraction buffer?
Most proteins contain free thiol groups which can undergo oxidation forming inter- and intramolecular disulfide bridges, these can be broken by the use of betamercaptoethanol in the buffer.
48
Why would we add an enzyme inhibitor to a extraction buffer?
Once the cell is disrupted, the organisational integrity of the cell is lost, and proteolytic enzymes that were carefully packaged and controlled within the intact cells are released, for example from lysosomes that may degrade proteins in the extract, including the protein of interest.
49
Why would we add an enzyme substrate and cofactors to a extraction buffer?
1. The substrate (included in the extraction buffer) binds to the active site of the enzyme (target) can stabilise the enzyme during purifi cation processes.
2. In the case of cofactors, they way be added to maintain enzymatic activity, so they can be detected when column fractions are screened.
50
Why would we add an EDTA to a extraction buffer?
1) To remove divalent metal ions by chelation that can react with thiol groups in proteins giving thiolates.
2) The sequestration of free magnesium ions by EDTA decreases the activity of any enzyme that uses ATP as a cofactor.
3) also reduces the activity of metalloproteases
51
Why would we add an Phosphatase inhibitor to a extraction buffer?
commonly used in buffers for protein kinase assays
52
What two classes of membrane proteins are there?
1. Extrinsic – these hydrophilic proteins are bound only to the surface of the cell, normally via electrostatic and H-bonds
2. Intrinsic – contain significant regions of hydrophobic amino acids embedded in the membrane, and associated with lipids, and have low solubility in aqueous buffer systems
53
How does one extract extrinsic membrane proteins?
Raising ionic conc. of extraction buffer or by either basic or acidic conditions.
54
How does one extract intrinsic membrane proteins?
With a buffer containing detergents
55
Name a non-ionic detergent?
Triton X-100
56
Name a ionic detergent?
SDS
57
What is the key difference between detergents and chaotropic agents?
Detergents denature proteins bases on solvating hydrophobic residues while chaotropic agents denature proteins based on weaking the hydrophobic effect.
58
Once membrane proteins are extraxted, why is it important to include a detergent in buffer before purification using chromatographic techniques?
To maintain protein solubility, The level of detergent used is normally 10- to 100-fold less than that used to extract the protein, in order to minimise any interference of the detergent with the chromatographic process.
59
By the use of sonification the sound waves cause disruption of cells by?
Shear force and cavitation
60
The sonification method is suitable for rel. small volumes of __ - __ cm3
What’s the efficiency of cell lysis by sonification?
1. 50
2. 100
3. 50-60 %
61
Is sonification a more gentle method than Repeated freeze-Thaw method for bacterial cells?
No
62
What cell disruption method is ideal for mammalian and plant tissue by shear force?
Blenders
63
What cell disruption method is excellent for microbial cells?
Presses
64
The initial extract, produced by the disruption of cells and tissue is refered to as?
Homogenate
65
In order to make the homogenate clear, we need to remove cell debris, how? (2 tissue and fat).
And if the solution is still cloudy by organelles and membrane fragments that’re way to small, we can make use firstly by?
1. Filtering by cheesecloth and 5000xg centrifugation
2. precipitation using celite, flocculants and the resultant precipitate is removed by 20000xg (45 min)
66
Why do we have to put attention to macromolecules in the cleared (removal of cell debris) homogenate? And how can these macromolecule be removed (3)
1. In the case of bacterial extracts they may be inherantly viscous, thus extremely difficult to centrifuge to produce a supernatant extract
2. DNase I, RNase, protamines
67
For bacterial extracts how do we remove carbohydrate capsular gum?
lysozyme
68
What does the distribution coefficient describe?
The way in which a compound(analyte) distributes between two immiscible phases
69
The resolution of a mixture of analytes ____ as the particle size of the stationary phases decreases, but such a decrease leads to a high back-pressure from the eluent flow. This is addressed by _____.
1. increases
2. HPLC
70
The most common method of sample introduction is by the use of a?
Loop injector
71
Explain isocratic/stepwise/gradient elution modes in zonal development?
1. Isocratic – when the composition of the mobile phase is constant
2. Gradient – In addition to the statement above if instead gradualy change the composition over time wrt. pH, salt conc. or polarity
72
What is the separation factor in liquid chromatography?
Is expressed as the selectivity of the system that, in turn, is a measure of its inherent ability to discriminate between two analytes
73
How can retention factor be used?
to compare the behaviour of an analyte in different chromatographic systems.
74
The smaller the plate height (the larger the value of N ), the ____ the analyte peak.
A number of processes oppose the formation of a narrow analyte peak, thereby _______the plate height.
1. smaller
2. increasing
75
Does plate height determine the width of the analyte?
Yes
76
What processes are responsible for peak broadening?
1. Longitudal diffusion
2. Multiple pathways
3. Eqilibration time between two phases
77
What is the van Demter equation?
The precise relationship between the 3 factors shown above and plate height is expressed by the equation.
78
What phenomenon counteracts the improvement in resolution achieved by increasing the column length?
Since the value of standard deviation is proportional to the square root of t R , it follows that if the elution time increases by a factor of four, the width of the peak will double. Thus, the longer it takes a given analyte to elute, the wider will be its peak.
79
What are the most common cause of fronting?
Column overloading
80
What is a probable explanation for tailing?
retention of analyte by a few sites (frequently hydroxyl groups) on the stationary phase, commonly on the inert support matrix. Such sites strongly adsorb molecules of the analyte and only slowly release them
81
Give the name of the process by which a reagent removes the sites of hydroxyl ground that are on the stationary phase, i.e. the inert support matrix?
capping
82
Unresolved peaks are refered to as?
Fused peaks
83
Why is Ion-exchange chromatography resins typically used in the earlier stages of a purification process?
Because of its high capacity its ability to measure the amount of material can more readidly be resolved into its components without causing peak overlap or fronting.
84
What does conjugation-activated agarose allow for?
immobilisation of suitable small and macro-molecules
85
Does affinity chromatography frequently used with protein fusion constructs?
Yes most frequently
86
Is Ion Exchange Chromatography considered a gentle purification method?
yes
87
In SEC what does the separation process depend on?
size
88
Why does SEC have limited resolving power?
Because it can practicaly resolve seperations between small and large proteins
89
A major advantage of SEC is that it’s gentle on proteins, that’s why it’s used as a ___ stage in preparations destined for protein crystallography and other applications that require functional protein
final
90
Besides size differences what other determinant can be calculated with SEC?
Rel. molecular mass
91
Why is SEC frequently used for desalting of protein solutions?
owing to the large size difference between inorganic ions and proteins
92
Why should the conc. of the analyte be as high as possible?
To get better resolution by mitigating diffusion
93
What type of group is bonded to agarose support matrix in HIC?, and is it strongly hydrophilic?
1. Phenyl is one of them
2. no
94
How are proteins eluted in HIC?
by applying a decreasing salt gradient
95
Up to 24 hours how should proteins be stored?
4 celcius, and 100 mM monovalent salt
96
For longer periods of time, how should protein be stored?
1. Below 0 degrees celcius
2. dialysed into final use buffer
3. snap-frozen in aliquots + glycerol
or freeze dry creating a lyophilised pellet
