Kap 5: Preparative protein biochemistry

Question 1

On average how many different fractionation steps are needed to purify a protein

Answer 1

4

Question 2

Protein purification can be broadly divided into ___ steps after the initial extraction
from the cellular source

Answer 2

3:
Capture
Intermediate stage to remove bulk impurities
polishing to achieve highly pure proteins

Question 3

What is the only truly accurate method to measure concentration of proteins

Answer 3

acid hydrolysis of a portion of the sample then do amino-acid analysis of the hydrolysate

Question 4

The acid hydrolysis method is time consuming, so we have reasonably quicker methods, most of these fall under !

Answer 4

calorimetric methods: reagent + protein = colored product

Question 5

What is the subsequent step after use of calorimetric method

Answer 5

then the colored product is measured spectrophotometrically and observed absorbance related to the amount of protein by calibration

Question 6

spectrophotometric methods regarding standard calibration curve, which calibration compound is common to use in colorimetric assay?

Answer 6

BSA

Question 7

why are calorimetric methods not absolute?

Answer 7

because the development of color is partly dependent on the amino-acid composition

Question 8

Name 3 protein concentration asseys!

Answer 8

UV (280 nm)
BCA (562 nm)
Bradford (595 nm)

Question 9

Ism the UV absorption essey destructive?

Answer 9

no

Question 10

How many times lower does UV absorption have in sensitivity comapred to BCA?

Answer 10

20x lower.

Question 11

In UV absorption essey why do we use a 260 nm/280nm ratio?

Answer 11

because nucleic acids absorb at the same wavelength 280 nm as aromatic amino-acid side chains of proteins, therfore we have a correction factor that tell us how clean the sample is from nucleic acid impurities.

Question 12

Describe BCA method [protein concentration esseay)

Answer 12

depends on the conversion

of Cu 2+ to Cu + under alkaline conditions. and complexation of cu+ with BCA –> purple color and absorbance at 562 nm

Question 13

What is the name of the binding-dye in bradford method?

At low pH, the free dye has absorption maxima at ___ and ___ nm

but when bound to protein this absorption is observed at?

The practical advantages of the method are that:

Why is this method considered relative ?

Answer 13

Coomassie Brilliant Blue
470
650
595
regent is simple to prep and the color develops rapidly and is stable.
becuase the amount of dye bidning very with the concentration of the basic amino-acids arginine & lysine in the protein

Question 14

What are the two problems with Bradford method?

Answer 14

1. that its a relative method wherby the binding of the dye
   varies with concentration of the basic amino acids
   arginine and lysine in the protein
2. many proteins will not dissolve properly in the acidic reaction medium

Question 15

cloning for protein synthesis using genetic engineering methodology is carried out using ____ methods

Answer 15

recombinant

Question 16

the manipulation of the gene of interest to engineer particular protein constructs is the ___ important step in the design of a protein purification procedure.

Answer 16

first

Question 17

5.3 ENGINEERING PROTEINS FOR PURIFICATION
5.3.1 Fusion Constructs to Aid Protein Purification
using a ___ at the N- or C-terminal ends provide means for the fusion protein to be selectively purified from the cell extract by __ ____

Answer 17

Tag

affinity chromatography

Question 18

5. 3 ENGINEERING PROTEINS FOR PURIFICATION
   
   5. 3.1 Fusion Constructs to Aid Protein Purification

Tag –protease site (peptide linkage to be enzymatically cleaved)—____

Answer 18

protein

Question 19

5. 3 ENGINEERING PROTEINS FOR PURIFICATION
   
   5. 3.1 Fusion Constructs to Aid Protein Purification

the poly-His-rag confers binding to:

Low concentrations of imidazole (10–20mM) may
be included in the wash buffer to increase ___ of the final eluate.

Answer 19

immobalized bivalent nickel or cobalt ions

purity

Question 20

5.3 ENGINEERING PROTEINS FOR PURIFICATION

As discussed above, it is possible to create fusion proteins by engineering the DNA
to contain the required sequence with a poly-His-tag for example. However, it may be desirable to introduce post translational modifications that cannot be achieved through molecular cloning or reliably achieved through chemical or enzymatic modification. In a process known
as ____ ___ ___.

Answer 20

expressed protein ligation

Question 21

5. 3 ENGINEERING PROTEINS FOR PURIFICATION
   
   5. 3.4 Secreted Proteins

number of advantages in manipulating the gene to ensure that the protein product is secreted from the cell, name 3!

Answer 21

1. To facilitate purification rather than rupturing cells to release the protein wherby other intracellular proteins would “contaminate”
2. Prevention of intracellular degradation of the cloned protein becuase cloned protein is recognised as foreign thus can be degraded by proteases, so Secretion of the protein into the culture medium should minimise this degradation.
3. Reduction of the intracellular concentration of toxic proteins by secretion to prevent cell death because the cell has a limit to the amount of protein to produce

Question 22

5.4 PRODUCING RECOMBINANT PROTEIN
With bateria with the example of E.coli, after the discovery of the lac operon, non-metabolisable analogues of lactose such as IPTG, that relieve _____ of the lac
repressor, were exploited to drive transcription and translation of the desired gene in
Escherichia coli . The main advantage is the relative ease to manipulate, transform and induce bacteria to produce a protein of interest.

Answer 22

repression

Question 23

Bacteria can be classified as either:

Answer 23

Gram positive or negative

Question 24

For Bacteria, whats the difference with gram negaitve to positive

Answer 24

the thinner layer of peptidoglycan is compensated with a second outer membrane layer of lipopolysaccharide

Question 25

For bacteria when the expression vector is transformed, there’re two ways to perform this, which two?

Answer 25

heat shock

short pulse of electricity ( electroporation)

Question 26

What is the reason for lack of expression of recombinant genes?

so how can one analyze the codon usage?

Answer 26

1. due to the sequence of amino acids containing signifi
   cant secondary structure that inhibit correct protein
   folding
2. Also, the gene of interest may utilise rare codons that
   exist in the source organism, but do not occur in the
   bacterial expression organism.

with bioinformatics

Question 27

Regarding protein expression what arw the three thing to look for in order for expression to be succesful?

Answer 27

1. if the starting material contains a high percentage of
   the over-expressed protein in relation to the total
   protein mixture. whereby one want to be at OD600 = 0.5 -1.0 to avoid cellular stress.
2. choice of bacterial strain becuase even small amount
   of expressed protein may induce cellular stress due to basal expression of the T7 RNA polymerase.
   a) that contains plasmids encoding for rare codons
   b) strong repressor function to avoid basal
   expression and are highly competent (i.e. easy to
   transform).
3. Oxygenation

Question 28

When proteins have been directed towards inclusion bodies what happens with the protein?

Answer 28

becomes insoluable.

Question 29

What is the most common media to culture bacteria for protein over-expression?

What is it composed of?

Answer 29

LB

2:1:1 tryptone, yeast extract and NaCl

Question 30

Before one can grow large cultures, the transformed bacteria are grown in an enriched medium
called ___ that allows viable bacteria to grow before plating out

Answer 30

SOC similar to LV but with different ratios of nutrients and is supllmented with: KCl, MgCl 2 , MgSO 4
and glucose.

Question 31

There is a deliberate absence of activating sugars (glucose) in LB in order to prevent basal expression of the plasmid, so that ____ can be added to cause protein expression.

Answer 31

IPTG

Question 32

Is it easier to disrupt yeast & fungi cells than mammalien cells

Answer 32

no

Question 33

By ranking which cell type is in the middle regarding cell rigidity:

1. Yeast and fungi cells
2. Plant cells
3. Mammalian cells

Answer 33

3

Question 34

going from recombinant expression vectors into cells, has many advantages therby the starting material will most likely be from one kind of cell culture

but post-translational modifications, poor folding or poor induction characteristics can still make it necessary
in some instances to purify native proteins, what is the initial extract in protein purification procedures?

Answer 34

using disruption methods to release the protein content of the cells into an appropriate buffer, unless the protein
is secreted.

Question 35

regarding extraction buffers what are the factors to make the solution comparable to conditions found inside cells?

Answer 35

a) ionic strength of 0.1–0.2M of a monovalent
salt
b) pH between 7 and 8,

Question 36

What are the two most common extraction buffers?

Answer 36

TRIS and phosphate buffers

Question 37

Regarding an extraction buffer for example TRIS, what are the additional range of other reagents to be added for specific purpose?

Answer 37

An antioxidant
reducing agent –> beta-mercaptoethanol to prevent
oxidation of free thiols (cysteine) thereby destrying
disupfide bridges ecuase th buffer has a oxididising
enviroment wheras within the cell its fairly reducing
enviro.
Enzyme inhibitors
are needed when cell is disrupted where proteolytic
enzymes now are relased that degrade proteins. also
to prevent protelysis one should perform at 4 *C

Enzyme substrate and cofactors
a) substrates that bind to enzyme active site to stabilize
the ezyme during purification process
b) cofactors that maintain enzymatic activity

Phosphatase inhibitors

EDTA
chelate divalent metal cations to mitigate binding to thiol groups in proteins and also decreases the activity of any
enzyme that uses ATP as a cofactor

PVP (absorbs phenolic compounds to mitigate binding to proteins

Sodium azide (for storing)

Question 38

5.5 CELL-DISRUPTION METHODS
5.5.1 Cell Disruption and Production of Initial Crude Extract
Membrane Proteins

Membrane-bound proteins are not relased by simple cell-disruption procedures.

1. Extrinsic type (hydrophilic) in nature: by increase of ___
   concentrations or acidic/basic conditions (pH 3-5 / pH
   9-12)
2. ____ type (embedded in membrane): by detegents like the ionic detegent: SDS or non-ionic –> Triton X-100.

Once extracted, intrinsic membrane proteins can be purifi ed using conventional chromatographic techniques such as size-exclusion, ion-exchange or affinity chromatography (using lectins).

Answer 38

ionic

Intrinsic

Question 39

Sonification (cell lysis technique)
ultrasound frequency at 30-__ s.
disruption of cells by shear force and ___
This method is suitable for relatively small volumes (50–__cm^3 )
rosette cell
The efficiency of cell lysis by sonication is thought to be around __–60%.

ideal for a suspension of cultured cells or ___ cells

Answer 39

60
cavitation
100
50
microbial

Question 40

blenders are ideal for disrupting ____ or plant tissue
How long should you blend?
This method is inappropriate for ___ and ___

Answer 40

mammalian
1 min
bacteria
yeast

Question 41

Presses (homogenizers)

excellent means for disrupting ____ cells

Answer 41

microbial

Question 42

Enzymatic methods for cell disruption

The peptidoglycan cell wall can therefore be removed from Gram-____bacteria with lysozyme, and if carried out in a suitable buffer, the cell membrane will rupture, owing to the ___ effect of the suspending buffer.

Gram-negative bacteria can similarly be disrupted by lysozyme, but treatment with ___, and if carried out in a ___ medium one can relase proteins from the periplasmic space without ruptering cell membrane

___ can be similarly disrupted using enzymes to degrade the cell wall, followed by either osmotic shock or mild physical force to disrupt the cell membrane.

Answer 42

positive
osmotic
EDTA
Yeast

Question 43

after homogenisation or lysis we get an initial extract that is referred to as a ____ or ____

Answer 43

homogenate

lysate

Question 44

If the homogenate contains small fragments of non-homogenised tissue from mammalian cell mixture, how can we remove those?

And what if we see fat floating, how to be removed?
if the solution is still cloudy one can!

Answer 44

with low speed (5000xg) centrifugation or double layer filtering (crude separation)

coarse filtration through glass wool or cheesecloth

precipitation then 20 000xg centrifugation 45 min

Question 45

Even the cleared homogenate contains not only proteins, but also other molecules such as DNA, RNA, carbohydrate and lipid, as well as any number of small-molecular-weight metabolites, which two way are there to remove these?

but in the case of unwanted macromolecules, what can be addeed to solution?

For bacterial extracts, carbohydrate capsular gum can be removed with

Answer 45

dialysis or size fractionation

DNase (reduces inital extract viscocity) or Rnase
or protamines – bind to DNA in the sperm head
or polyethyleneimine:
binds to the phosphate groups in nucleic acids, and is.
very effective, precipitating DNA and RNA almost
instantly.

lysozyme

Question 46

Partition coefficient in liquid chromatography

what does it describe?

Answer 46

the the way the analyte distributes between two immiscible phases

Question 47

liquid chromatography

The resolution of a mixture of analytes increases as the particle size of the stationary
phases decreases, but such a decrease leads to a high back-pressure from the eluent
flow when one uses a pumping system. This is addressed by ____

The application of samples onto HPLC columns in the correct way is a particularly important factor in achieving successful separations. The most common method of
sample introduction is by use of a __ ___

Answer 47

HPLC

loop injector

Question 48

liquid chromatography

Column chromatographic techniques can be subdivided on the basis of the development and elution modes into:

Answer 48

zonal development

affinity development

Question 49

Regarding Column chromatographic techniques using the zonal development, describe gradient elution

Answer 49

a step-wise or continous change in mobile phase composition with respect to pH or salt conc. or polarity

Question 50

Regarding Column chromatographic techniques using the affinity development, is it practically useful for gas chromatography?

The analytes bind to the stationary phase with a strength determined by their affinity constant for the phase. The
analytes are then selectively eluted by using a mobile phase containing a specific solute that has a ___ affinity for the stationary phase than have the analytes in
the sample.

The one with ___ affinity is being eluted first

Answer 50

no
higher
lowest

Question 51

Two general factors affect the behavior of each analyte, which two?

Answer 51

1. basic mechanisms defining the chromatographic
   process, i.e choice of chromatography type method
   which involve the unique kinetic and thermodynamic
   processes that characterize the interaction of each
   analyte with the stationary phase
2. diffusion (broadening) and non-specific interactions (tailing)

Question 52

What is the dead time?

What is adjusted retention time?

the retention time is composed of two components, which two?

Answer 52

the time at which analytes pass thrue the free spaces between the particles of the matrix coated with the stationary phase., will be same for all analytes

the time the stationary phase retains the analyte

the above

Question 53

What is retention factor?

if the analyte spends an equal time in the
stationary and mobile phases, its tR would equal __× tM and its k would thus be __

Answer 53

the additional time that the analyte takes to elute from the column relative to an unretained or excluded analyte
that does not interact with the stationary phase

2
1

Question 54

The retention factor is related to the distribution coeffi cient of the analyte therby giving us a relative _____ of the analyte between the two phases.

Answer 54

concentrations

Question 55

What does the volymetric phase ratio tell us

Answer 55

it describes a relation between concentration and volume, if we increase the volume of the analyte on the stationary phase the retention faktor will increase therby increasing the ultion time and ditribution coefficient between the two immicible phases.

Question 56

What does the seperation factor tell us?

Answer 56

a measure of its inherent ability to discriminate between two analytes, which is the relative retention ratio for the two phases

Question 57

Plate height is simply related to the ___ of the analyte peak

Answer 57

width

Question 58

The smaller the plate height (the larger the value of N ), the _____ the analyte peak

Answer 58

narrower

Question 59

What is the van Deemter equation?

Answer 59

It incorporates effcts of the column chromatography techniqye where,

a) longitudual diffusion is reflected with Fick’s law of
diffusion that states that an analyte will diffuse from a
region of high conc to low conc at a rate determined
by. the conc. gradient between the two phasea and the
diffusion coefficient for the analyte – therby invesrily
proportinal with flow rate and an analyte with narrow
band will diffuse out –> band broadening
b) equilibration time due to the partitioning of the analyte
renders analyte band spreading and is therfore directly
proportional to flow rate

these two factors together with the multiple pathway dilemma affect the plate height value –> determines the peak width

the these 3 factors vs. plate height is the equation.

Question 60

the width of an analyte peak is expressed in terms of the standard deviation, which is half the peak width at the point of inflection, and since it is proportional to the square of tR, thus if we elution time is increased by a factor of four, the width of the peak will ___

Thus, the longer it takes a given analyte to elute, the ___ will be its peak

Answer 60

double

wider

Question 61

The most common cause of fronting (–> asymmetric non-ideal Gaussian peak) is:

In cases where the rise in the peak is normal, but the tail is protracted, the phenomenon is known as ___

Answer 61

overloading the column

tailing

Question 62

What is the probable explanation of tailing?

how can it be overcome?

Answer 62

is the 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

by chemically removing the sites, for example by treating the matrix with a silanising reagent such as hexamethyldisilazine. This process is sometimes referred to as capping

Question 63

Unresolved peaks are referred

to as ___ peaks

Answer 63

fused

Question 64

What three thing in column chromatography influenses resolution?

Answer 64

column efficiency, selectivity factors (see Equation 5.7)

and retention factors

Question 65

The ____ of a particular chromatographic separation is a measure of the amount of material that can be resolved into its components without causing peak overlap or fronting.

Answer 65

capacity

Question 66

In affinity chromatography how can one elute bound protein of intrest? (3 ways)

Answer 66

with changing pH or increasing salt strength or passing through a high concentration of unbound free ligand.

Question 67

What seperates proteins with ion-exchange chromatography?

where in the purification step process is this method best suited

Answer 67

their their proportions of charge amino acids, thus posseses different net charges at a particular pH

in the beginning. Or as a ‘polishing’ step after a certain purity has been achieved because it is a relatively gentle
purification method.

Question 68

With gel filtration the beads have different pore sizes and therefore slightly differing amounts of cross-linking

So what is the main use of this method regarding portein size?

in practicality why could dit be used?

Why should the concentration of the analyte be as high as possible?

why is it important to choose the appropriate column size?

Answer 68

to isolate or resolve a protein of interest that is either large or small giving better resolving power. is often used as a final stage in preparations destined for protein crystallography and other applications that
require functional protein

it is frequently used for the de-salting of protein solutions in order to determine the relative molecular mass of a protein

in order to have less diffusion.

because it’s necessary to have a equilibrated system where the amount of material don’t get lost due to dilution and surface interactions.

Question 69

Name the method that is stability-based for purification

Answer 69

denaturing fractionation

Question 70

What does denaturing fractionation exploit

Answer 70

heat sensitivities that depict the ability to disrupt the tertiary force interactions thereby one can use thermal denaturation as the method for the denaturation fractionation in line with purification.

Question 71

Describe Solubility-Based Purification

Answer 71

Precipitated native protein by aggregation due to addition of organic solvents or neutral salts; an effect due to difference in solubility regarding proteins depending on their charge, polarity, and hydrophobic ratios.

Question 72

In Solubility-Based Purification does the native protein denature during this process?

Answer 72

no

Question 73

What salt is frequently used for salt fractionation?

when increasing the concentration of salt what happens with water?

but if we replace salt with organic solvent what happens with water?

Answer 73

ammonium sulfate

water is forces to interact with the hydrophobic parts of the protein, due to this dilemma the proteins will aggregate and precipitate out of solution due to the fact that water cant solvate the proteins enough.

the water is diluted out by hydrating the organic solvent molecules thereby reducing the dielectric constant because the water molecules don’t interact with the charged and polar groups on the proteins. –> thereby more and more hydrophobic parts get exposed –> aggregation –> precipitation, in decreasing order of the number of charged groups on their surface as the
organic solvent concentration is increased.

Question 74

Describe Isoelectric Precipitation Fractionation!

Answer 74

by changing pH to the isoelectric point of a specific protein, the protein in question will have minimum solubility allowing proteins to actually approach each other where opposite charges on proteins to interact forming aggregates

Question 75

Describe Inclusion Body Purification!

Answer 75

inclusion bodies are insoluble particles of partially or incorrectly folded proteins aggregations, these aggregations can be separated with centrifugation then solubilized and denatured with a chaotropic agent in the presence of a reducing agent to disrupt disulfide bridges

for refolding of the denatured protein one can use dilution or dialysis against a suitable buffer to attain the active (native) conformation.

Question 76

5.10 MONITORING PROTEIN PURIFICATION

which is the most common method to analyze the identity of proteins in aliquots of each fraction?

Answer 76

SDS PAGE

Question 77

5.10 MONITORING PROTEIN PURIFICATION

what method is used for identifying proteins having a corresponding antibody available

Answer 77

dot blot method, where one dry the samples onto a nitrocellulose

or use immunoassay or radioimmunoassay

Question 78

5.10 MONITORING PROTEIN PURIFICATION
For recombinant proteins that are expressed as a fusion protein, i.e. linked to a tag that aids purification by an affi nity step, how is it possible to asses the identity of a protein of intrest?

What is the alternative way?

Answer 78

by evaluating the binding and relase to/ffrom the affinity resin.

with a 2nd protein that can be easily essayed using calorimetric assay. -galactosidas

Question 79

5.10 MONITORING PROTEIN PURIFICATION
if the target protein is an enzyme one can use __ __ to identify a protein of intrest!

What is acceptable yield?

Answer 79

enzymatic activity. becuase the actual measurement relies on kinetic concepts there’re suitable analytical procedures avaliable (CHPT 23)

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