Kap 5: Preparative protein biochemistry Flashcards
On average how many different fractionation steps are needed to purify a protein
4
Protein purification can be broadly divided into ___ steps after the initial extraction
from the cellular source
3:
Capture
Intermediate stage to remove bulk impurities
polishing to achieve highly pure proteins
What is the only truly accurate method to measure concentration of proteins
acid hydrolysis of a portion of the sample then do amino-acid analysis of the hydrolysate
The acid hydrolysis method is time consuming, so we have reasonably quicker methods, most of these fall under !
calorimetric methods: reagent + protein = colored product
What is the subsequent step after use of calorimetric method
then the colored product is measured spectrophotometrically and observed absorbance related to the amount of protein by calibration
spectrophotometric methods regarding standard calibration curve, which calibration compound is common to use in colorimetric assay?
BSA
why are calorimetric methods not absolute?
because the development of color is partly dependent on the amino-acid composition
Name 3 protein concentration asseys!
UV (280 nm)
BCA (562 nm)
Bradford (595 nm)
Ism the UV absorption essey destructive?
no
How many times lower does UV absorption have in sensitivity comapred to BCA?
20x lower.
In UV absorption essey why do we use a 260 nm/280nm ratio?
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.
Describe BCA method [protein concentration esseay)
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
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 ?
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
What are the two problems with Bradford method?
- 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 - many proteins will not dissolve properly in the acidic reaction medium
cloning for protein synthesis using genetic engineering methodology is carried out using ____ methods
recombinant
the manipulation of the gene of interest to engineer particular protein constructs is the ___ important step in the design of a protein purification procedure.
first
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 __ ____
Tag
affinity chromatography
- 3 ENGINEERING PROTEINS FOR PURIFICATION
- 3.1 Fusion Constructs to Aid Protein Purification
Tag –protease site (peptide linkage to be enzymatically cleaved)—____
protein
- 3 ENGINEERING PROTEINS FOR PURIFICATION
- 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.
immobalized bivalent nickel or cobalt ions
purity
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 ____ ___ ___.
expressed protein ligation
- 3 ENGINEERING PROTEINS FOR PURIFICATION
- 3.4 Secreted Proteins
number of advantages in manipulating the gene to ensure that the protein product is secreted from the cell, name 3!
- To facilitate purification rather than rupturing cells to release the protein wherby other intracellular proteins would “contaminate”
- 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.
- 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
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.
repression
Bacteria can be classified as either:
Gram positive or negative
For Bacteria, whats the difference with gram negaitve to positive
the thinner layer of peptidoglycan is compensated with a second outer membrane layer of lipopolysaccharide
For bacteria when the expression vector is transformed, there’re two ways to perform this, which two?
heat shock
short pulse of electricity ( electroporation)
What is the reason for lack of expression of recombinant genes?
so how can one analyze the codon usage?
- due to the sequence of amino acids containing signifi
cant secondary structure that inhibit correct protein
folding - 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
Regarding protein expression what arw the three thing to look for in order for expression to be succesful?
- 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. - 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). - Oxygenation
When proteins have been directed towards inclusion bodies what happens with the protein?
becomes insoluable.
What is the most common media to culture bacteria for protein over-expression?
What is it composed of?
LB
2:1:1 tryptone, yeast extract and NaCl
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
SOC similar to LV but with different ratios of nutrients and is supllmented with: KCl, MgCl 2 , MgSO 4
and glucose.
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.
IPTG
Is it easier to disrupt yeast & fungi cells than mammalien cells
no
By ranking which cell type is in the middle regarding cell rigidity:
- Yeast and fungi cells
- Plant cells
- Mammalian cells
3
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?
using disruption methods to release the protein content of the cells into an appropriate buffer, unless the protein
is secreted.
regarding extraction buffers what are the factors to make the solution comparable to conditions found inside cells?
a) ionic strength of 0.1–0.2M of a monovalent
salt
b) pH between 7 and 8,
What are the two most common extraction buffers?
TRIS and phosphate buffers
Regarding an extraction buffer for example TRIS, what are the additional range of other reagents to be added for specific purpose?
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)
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.
- Extrinsic type (hydrophilic) in nature: by increase of ___
concentrations or acidic/basic conditions (pH 3-5 / pH
9-12) - ____ 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).
ionic
Intrinsic
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
60 cavitation 100 50 microbial
blenders are ideal for disrupting ____ or plant tissue
How long should you blend?
This method is inappropriate for ___ and ___
mammalian
1 min
bacteria
yeast
Presses (homogenizers)
excellent means for disrupting ____ cells
microbial
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.
positive
osmotic
EDTA
Yeast
after homogenisation or lysis we get an initial extract that is referred to as a ____ or ____
homogenate
lysate
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!
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
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
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
Partition coefficient in liquid chromatography
what does it describe?
the the way the analyte distributes between two immiscible phases
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 __ ___
HPLC
loop injector
liquid chromatography
Column chromatographic techniques can be subdivided on the basis of the development and elution modes into:
zonal development
affinity development
Regarding Column chromatographic techniques using the zonal development, describe gradient elution
a step-wise or continous change in mobile phase composition with respect to pH or salt conc. or polarity
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
no
higher
lowest
Two general factors affect the behavior of each analyte, which two?
- 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 - diffusion (broadening) and non-specific interactions (tailing)
What is the dead time?
What is adjusted retention time?
the retention time is composed of two components, which two?
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
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 __
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
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.
concentrations
What does the volymetric phase ratio tell us
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.
What does the seperation factor tell us?
a measure of its inherent ability to discriminate between two analytes, which is the relative retention ratio for the two phases
Plate height is simply related to the ___ of the analyte peak
width
The smaller the plate height (the larger the value of N ), the _____ the analyte peak
narrower
What is the van Deemter equation?
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.
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
double
wider
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 ___
overloading the column
tailing
What is the probable explanation of tailing?
how can it be overcome?
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
Unresolved peaks are referred
to as ___ peaks
fused
What three thing in column chromatography influenses resolution?
column efficiency, selectivity factors (see Equation 5.7)
and retention factors
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.
capacity
In affinity chromatography how can one elute bound protein of intrest? (3 ways)
with changing pH or increasing salt strength or passing through a high concentration of unbound free ligand.
What seperates proteins with ion-exchange chromatography?
where in the purification step process is this method best suited
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.
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?
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.
Name the method that is stability-based for purification
denaturing fractionation
What does denaturing fractionation exploit
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.
Describe Solubility-Based Purification
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.
In Solubility-Based Purification does the native protein denature during this process?
no
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?
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.
Describe Isoelectric Precipitation Fractionation!
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
Describe Inclusion Body Purification!
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.
5.10 MONITORING PROTEIN PURIFICATION
which is the most common method to analyze the identity of proteins in aliquots of each fraction?
SDS PAGE
5.10 MONITORING PROTEIN PURIFICATION
what method is used for identifying proteins having a corresponding antibody available
dot blot method, where one dry the samples onto a nitrocellulose
or use immunoassay or radioimmunoassay
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?
by evaluating the binding and relase to/ffrom the affinity resin.
with a 2nd protein that can be easily essayed using calorimetric assay. -galactosidas
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?
enzymatic activity. becuase the actual measurement relies on kinetic concepts there’re suitable analytical procedures avaliable (CHPT 23)
<70 %
