分子生物學
If you take this course, you will be very busy during this semester. You have lots of home works to do. If you can not face challenges, please do not take this course.
Biotech Lab protocols: http://www.lab-manual.com/
Let’s watch this movie:
http://www.pbs.org/wgbh/nova/genome/program.html#
Mendel’s Discoveries
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookgenintro.html
An
Austrian monk, Gregor Mendel, developed the fundamental principles that would
become the modern science of genetics.
Principle of Independent
Segregation
According to the principle of segregation,
for any particular trait, the pair of alleles of each parent separate and only
one allele passes from each parent on to an offspring. Which allele in a
parent's pair of alleles is inherited is a matter of chance.
Principle of
Independent Assortment
When gametes are formed in diploid
organisms, the segregation of each gene pair does not affect the segregation of
other gene pairs as long as the gene pairs are on separate chromosomes
Thomas Hunt Morgan
Chromosome Mapping
Three points cross:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/linkage/linkage3.htm
http://www.genetics.wisc.edu/courses/466/summer2003/threepoint/
Frederick Griffith’s Experiment
http://life.nthu.edu.tw/~rrandd/90s1/b881622/bioinfo.swf
Hershey and Chase Experiment
http://www.accessexcellence.org/RC/VL/GG/hershey.html
DNA Structure:(Download Chime http://www.mdl.com/my_account/register1.jsp
[aMfehTFg])
http://biotech-adventure.okstate.edu/low/basics/dna_structure/
1 hr
作業 1hr 10%
Matthew Meselson and Frank W. Stahl
http://www.accessexcellence.org/RC/VL/GG/possible.html
http://tidepool.st.usm.edu/crswr/meselsonstahl.html
DNA===>RNA(mRNA tRNA rRNA)
RNA Structure
http://www-scf.usc.edu/~chem203/resources/DNA/rna_structure.html
The Central Dogma
http://www.accessexcellence.org/RC/VL/GG/central.html
Transcription and Translation
Transcription:http://www.ncc.gmu.edu/dna/mRNAanim.htm
http://www.csuchico.edu/~jbell/Biol207/animations/transcription.html
http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html
http://edtech.clas.pdx.edu/gene_expression_tutorial/transcription.html
http://www.cat.cc.md.us/biotutorials/protsyn/transc.html
Translation:
http://www.ncc.gmu.edu/dna/ANIMPROT.htm
http://nobelprize.org/medicine/educational/dna/b/translation/translation_ani.html
Genetic Codes:
http://psyche.uthct.edu/shaun/SBlack/geneticd.html
http://www.accessexcellence.org/RC/VL/GG/genetic.html
http://www.people.virginia.edu/~rjh9u/code.html
http://web.indstate.edu/thcme/mwking/protein-synthesis.html
http://cellbio.utmb.edu/cellbio/ribosome.htm
The Direction of Protein Synthesis
NH2èCOOH
Equilibrium constant:
Calculator:
http://chemmac1.usc.edu/equilibrium/equilibrium.php
http://chemmac1.usc.edu/equilibrium/more_eq.php
http://grashof.engr.colostate.edu/tools/equil.html
Free energy
http://wine1.sb.fsu.edu/chm1045/notes/Gases/IdealGas/Gases04.htm
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch21/gibbs.html
Calculator:
http://www.shodor.org/UNChem/advanced/thermo/thermocalc.html
http://mc2.cchem.berkeley.edu/Java/Gibbs/Gibbs.html
http://members.aol.com/engware/calc1.htm
RNA Secondary Structure
http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi
Amino Acids and Protein Structure
http://www.otterbein.edu/home/fac/dnhjhns/chem220/tutorial3/tutorial3.html
http://www.escience.ws/b572/L9/L9.htm
http://info.bio.cmu.edu/Courses/BiochemMols/AAViewer/AAVFrameset.htm
http://www.accessexcellence.org/RC/VL/GG/prot_Struct.html
http://www.clunet.edu/BioDev/omm/chymo/chymo.htm
1 hr
作業 作業 1hr 10%
全班同學分工合作,將http://gslc.genetics.utah.edu/units/basics/protein/ 網頁改編為中文,並將file 寄至 yenlee@nttu.edu.tw 並註明每個人做了什麼。(請先看15 min 然後討論如何分工合作45 min,作業在2週內[28天]繳,逾期無分)
X-ray and NMR
Protein Structure Prediction
http://www.embl-heidelberg.de/predictprotein/predictprotein.html
http://restools.sdsc.edu/biotools/biotools9.html
http://www.russell.embl.de/gtsp/
Different Protein Functions Arise from Various Domain Combinations
|
Allosteric Regulation
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/protsyn/regulation/comp.html
DNA grooves
http://www.albany.edu/~achm110/bdnagrooveandwc.html
DNA Strands Can Separate and Reassociating
http://www.dnalc.org/Shockwave/southan.html
http://www.escience.ws/b572/L22/south.html
http://www.accessexcellence.org/RC/VL/GG/southBlotg.html
DNA OD Reading and Melting Temperature
http://www.xs4all.nl/~jori/concentratie.html
http://www.eden.rutgers.edu/~napoli/DNA-self.htm
http://www.basic.northwestern.edu/biotools/oligocalc.html
http://paris.chem.yale.edu/extinct.html
http://www.eden.rutgers.edu/~napoli/DNA-non.htm
DNA Linking Number
http://wine1.sb.fsu.edu/bch5425/lect07/lect07.htm
http://wine1.sb.fsu.edu/bch5425/lect08/lect08.htm
http://www.mol-biol4masters.com/Deoxy_Ribonucleic_Acid4-Super_Coiling.htm
http://www.rci.rutgers.edu/~molbio/Courses/MBB_408_512/Lec21.doc
Chromosome Structure
http://www.average.org/~pruss/nucleosome.html
http://www.accessexcellence.org/RC/VL/GG/nucleosome.html
http://biology.kenyon.edu/courses/biol114/Chap01/chrom_struct.html
1hr
(That is all for the 1st week.)
Experimnet 25hrs 20% (You have 4 weeks to complete this experiment and one week to prepare your presentation. Your presentation will be on the 7th week. )
Protocol modified from
http://homepages.gac.edu/~cellab/contents.html#chpt-5
http://gcg.tran.wau.nl/local/Biochem/educatio/BSM.htm
http://juang.bst.ntu.edu.tw/ECX/Con0.htm
Purification and characterization of proteins/enzymes:
At the end of the experiment, you should have:
some 'feeling' for working with proteins;
insight in the methods applied in protein purification;
practical experience in protein purification (each group of 3 – 4 students
isolates one protein/enzyme);
The score you obtain for this experiment is determined by:
the results obtained during your practical work (3/4) and your presentation(1/4).
Housekeeping rules
Each group receives basic equipment. Be careful with it.
Used glass-pipettes: rinse with water and place in glass container with
5% 沙拉脫
Used glasswares: rinse with water and place them in the original places
Keep (the environment of)
balances, pH-meters, spectrophotometers,…..clean
Remove everything when you are finished
Do not leave unmarked beakers with undefined solutions
After weighing, close bottles with chemicals and put it back
Clean waterbaths now and then
Proteins are stored in the refrigerator
Carefully when handle chemical waste
Be careful with cuvettes, especially quartz cuvettes
Evaluation of results
Results obtained during the experiment
will be discussed after you finish it. Each group will be asked to give a
presentation of the results they obtained.
During this presentation, each group should have available:
examples of the calculations made (for example, to calculate specific
activities);
all kinds of graphs made during the experiment (for example: calibration
graphs; graphs of enzyme activity at different pH and temperature; graphs to
calculate protein molecular weights from gel filtration and gel
electrophoresis, etc.);
You should have : introduction、materials and methods、results、discussions、and references,
in your presentation.
Your presentation is your final report and will be graded.
Protein/Enzyme purification
Why purify proteins
To understand what is going on in a cell and between cells:
reconstruction of metabolic and regulatory pathways. Pure enzymes/proteins
required to study reactions, kinetics, regulation, etc.
to understand deviations in normal metabolism or regulation processes,
etc., due to abnormal enzymes/proteins
to make a rational design of drugs possible, based on the 3D-structure
of a protein.
many proteins/enzymes have themselves an added value, as biocatalysts
(proteases, lipases, glucose isomerase), therapeutics (insulin, interleukins),
etc.
any many other reasons.
Degree of purity required depends on application
Keep structure and activity
intact
Physical boundaries of protein stability:
pH
temperature
salt concentration
oxygen sensitivity
storage
mechanical forces
How is purification measured ?
Determination of specific activity
Physical methods: SDS-PAGE;
gelfiltration
Preparing extracts for
purification
Sources -
animal tissues
plants
microorganisms
culture media
subcellular fractions (e.g. mitochondria, membranes)etc.
Recombinant DNA technology
is a very useful tool for protein purification
for 'overproduction' of proteins using expression vectors
for application of 'tags' to proteins
for excretion of proteins into the culture medium
Disruption and
homogenization of cells, tissue, etc.
Different techniques: grinding, sonication, French pressure cell
disruption, depending of cell types
Isolation of organelles; solubility of membranes;
Excreted proteins
Clearing of extracts
Cleared extracts should be used for column steps; clearing by
centrifugation or filtration; solubility of membranes
Protein stability
Temperature often 0 – 4o C (ice, cold
room)
pH determine pH where
enzyme
is most stable before
attempting purification;
work in buffers
Salt
Oxygen anaerobic conditions for
oxygen-labile enzymes
Proteolytic
enzymes add inhibitors: EDTA, benzamidine,
etc.
Storage frozen, suspension, addition
of
glycerol
Mechanical avoid foaming
forces
Buffers
buffering capacity
pKa
DpKa /DT
Henderson-Hasselbalch equation:
pH = pKa + log
(base / conjugated acid)
Main types of purification
methods
The following main types of purification methods for proteins can be
distinguished:
1. Precipitation methods
2. Separation based on molecular size
3. Separation based on charge
4. Separation based on specific interaction
with other biomolecules
5. Separation based on other principles
Examples:
1. Ammonium sulfate precipitation
2. Gel filtration
3. Ion-exchange chromatography
4. Bio-affinity chromatography
5. Hydrophobic interaction chromatography…..
stoichiometry of
participating reagents
requirement for cofactors
pH-optimum
temperature optimum
concentration of substrates
[S]
>> KM à v
~ [E]
continuous vs. discontinuous
assay
Alcohol
dehydrogenase:
NAD+
+ EtOH ----> NADH + aceetaldehyde
Calculation enzyme activity
Assay mix: 0.99 ml 0.1 M Tris/HCl, pH 9.0, 100 mM
EtOH, 0.5 mM NAD+,
Assay started by
addition of 0.01 ml ADH, 50x diluted from stock solution
Increase of
absorbance at 340 nm followed:
DA340 =
0.6/min.
à v = [ DA340/min ]/eNADH, 340 = 0.6/6.22 =
0.096 mM.min-1
à Amount of enzyme in assay
in Units:
0.096 mM.min-1 = 0.096 mmol.ml-1.
min-1 = 0.096 U.ml-1
à Amount of enzyme in ADH
stock solution:
0.096
x 1000/10 x 50 = 480 U.ml-1.
(dilution in (dilution prior
cuvet: 0.01 ml to addition to
enzyme + cuvet)
0.99 ml buffer)
Measure for each purification step:
1.
The volume of the enzyme solution (ml)
2.
The protein content of the solution (mg.ml-1)
3.
The activity of the enzyme solution (U.ml-1)
Total amount of enzyme (U):
Activity (U.ml-1) x volume (ml)
Specific activity (U.mg-1):
Activity (U.ml-1)
/ protein content (mg.ml-1)
Yield (%):
Total amount of enzyme after
a purification step / total amount of enzyme before that step
Purification factor:
Specific activity of enzyme after a
purification step / specific activity before that step
Purity-check: SDS-PAGE
Precipitation methods
Protein stability in
solution dependent on:
electrostatic interactions (ionogenic, amino acid side chains; salts)
H-bridges (sidechains; water)
hydrophobic interactions
Perturbation of interactions might cause precipitation
of proteins:
temperature
pH (dependent on pI)
salts
lipophylic agents (e.g.
ethanol)
cross-linking agents (e.g. protamine
sulfate)
water-extracting agents (e.g.
polyethylene glycol)
Why ammonium sulfate ?
Most proteins precipitate in saturated solution (4 M)
Low heat of solubilization (à prevents denaturation of proteins)
Low density of saturated solutions (1.25 g/cm3) à proteins can be collected
in pellets by centrifugation
Concentrated solutions prevent microbial growth
Protects most proteins from denaturation
Cheap
Applications ammonium
sulfate precipitation
Concentration of proteins by bulk
precipitation
Purification of proteins by fractionation
due to differences in solubility
Ammonium sulfate can be
added
as a solid: to be added to reach a saturation level from a saturated (=
100%, w/v) solution
Note: saturation level
is dependent on temperature
Pilot experiment ammonium
sulfate precipitation
1. 1 ml protein samples on ice
(appr. 10 samples)
2. Add solid ammonium sulfate to 0, 10%, 20%,
30%....90% saturation
Mix well, until a.s. is fully dissolved.
No vortexing.
3. Leave on ice (10 min.)
4. Centrifuge, 10 min. 13.000
rpm, cold room
5. Look for precipitates.
Transfer supernatants to clean tubes
and measure activity
This protein precipitates between 40%
and 70% ammonium sulfate saturation (‘fractionation range’).
x = 40%; y = 70% in this
example.
Preparative ammonium sulfate
fractionation
1. Measure volume of protein solution (M1).
Add ammonium sulfate to x% saturation , equilibrate, centrifuge.
2. Collect supernatant
(S1) and keep the pellet
(P1). Measure volume of
supernatant again, add ammonium sulfate to y% saturation , equilibrate,
centrifuge.
3. Collect supernatant (S2) and
pellet (P2).
4. Dissolve pellets P1 and P2 in
buffer.
5. Measure activity of supernatants (S1 and
S2) and dissolved pellets (P1, P2). P2 should contain the highest activity.
If the purpose of the precipitation is concentrating the enzyme, step 1
(saturation to x%) can be omitted.
Other precipitation methods
Polyethylene glycol
neutral, non-denaturating
compound
-
low heat of solution
-
steric exclusion mechanism (binds water)
Protamine sulfate
small, basic
proteins from sperm(many Arg and Lys residues)
precipitation of
large protein complexes (ribosomes), DNA, RNA by complexation
precipitation is
concentration dependent
Apolar solvents (acetone, alcohol)
low temperature
(- 5 OC)
denaturation of
proteins
Trichloroacetic acid
6% (w/v) final
concentration
unfolds/denaturates
proteins
Gel filtration
column chromatography
separates
biomolecules according to size
(hydrodynamic volume)
(size-exclusion
chomatography)
proteins do
not attach to column
stationary
phase: porous, cross-linked beads
(dextran,
agarose, polyacrylamide)
degree
of cross-linking determines diameter of pores and
fractionation range of biomolecules of
different size
Column parameters:
Vt = total
volume of packed column
(= height of column x cross-sectional
area, pr2)
V0 = void
volume
(free
accessible area, fully accessible for all molecules, independent of size)
Vi = internal
volume of b
eads
(only accessible for small molecules
+ solutes,
depending on size of pores and
molecules)
Vg = volume
of gel (solid material)
Vt = V0 + Vi (+Vg)
(Vg is very low for most materials and can be neglected)
Vt = V0 + Vi
The elution
volume Ve for a specific compound can be described as:
Ve = V0 + Kav Vi
with Kav the partition coefficient
which describes how much of the internal volume is available for the compound
(0 < Kav <1)
or:
Kav = (Ve
- V0) / (Vt
- V0)
Ve is determined by, e.g. activity measurements
V0
is measured by elution of blue dextran (Mr > 106)
Vt
is a) calculated (l . pr2)
b) determined
by elution of a small molecule (e.g. acetone)
Setup for gelfiltration
-
Classical setup for conventional soft gels (contains usually also
fraction collector)
Practice
Column
-Choice of column material: particle
size, fractionation range
De-aerated
buffers and column materials
Long columns
Column packing
Flow rates
during packing and running column
(cm/hour;
ml/hour)
Samples
-
Cleared
samples
-
Small
sample volumes (< 5% of column volume)
Solvent
Addition of
salt (appr. 0.2 M) to prevent electrostatic interaction
Addition of
stabilizing agents (protease inhibitors, antimicrobial agents, e.g. sodium azide)
Regeneration and storage
Regeneration
simple
Addition of
antimicrobial agents, ethanol
Fractionation
ranges
Name
medium
|
Type of matrix |
Particle sizeof hydratedbeads (mm) |
Fractionation range for globular proteins (Da) |
||||
Sephadex
|
Dextran |
|
|
||||
G-10 |
|
40 - 120 |
- 700 |
||||
G-25 medium |
|
50 - 150 |
100 - 5.000 |
||||
G-50 medium |
|
50 - 150 |
500 -
10.000 |
||||
G-75 |
|
40 - 120 |
1.000 -
50.000 |
||||
G-100 |
|
40 - 120 |
1.000 -
100.000 |
||||
G-150 |
|
40 - 120 |
1.000 -
150.000 |
||||
G-200 |
|
40 - 120 |
1.000 -
200.000 |
||||
|
|
|
|
||||
Sepharose CL |
Cross-linked agarose |
|
|
||||
6B |
|
45 - 165 |
10.000 -
4.000.000 |
||||
4B |
|
45 - 165 |
60.000 -
20.000.000 |
||||
2B |
|
60 - 200 |
70.000 -
40.000.000 |
||||
|
|
|
|
||||
Sephacryl
HR
|
Cross-linked allyldextran/ bisacrylamide |
|
|
||||
S-100 |
|
25 - 75 |
1.000 -
100.000 |
||||
S-200 |
|
25 - 75 |
5.000 -
250.000 |
||||
S-300 |
|
25 - 75 |
10.000 - 1.500.000 |
||||
S-400 |
|
25 - 75 |
20.000 - 8.000.000 |
||||
|
|
|
|
||||
Bio-Gel
P
|
Polyacrylamide |
|
|
||||
P-6DG |
|
90 - 180 |
1.000 - 6.000 |
||||
P-10 medium |
|
90 - 180 |
1.500 -
20.000 |
||||
P-30 medium |
|
90 - 180 |
2.500 -
40.000 |
||||
P-60 medium |
|
90 - 180 |
3.000 -
60.000 |
||||
P-100 medium |
|
90 - 180 |
5.000 - 100.000 |
||||
|
|
|
|
||||
Bio-Gel A |
Agarose |
|
|
||||
A-0.5m medium |
|
75 - 150 |
10.000 - 500.000 |
||||
A-1.5m medium |
|
75 - 150 |
10.000 - 1.500.000 |
||||
A-5m medium |
|
75 - 150 |
10.000 - 5.000.000 |
||||
A-15m medium |
|
75 - 150 |
40.000 - 15.000.000 |
||||
|
|
|
|
||||
Ultrogel |
Agarose |
|
|
||||
A6 |
|
60 - 140 |
25.000 - 2.400.000 |
||||
A4 |
|
60 - 140 |
55.000 - 9.000.000 |
||||
A2 |
|
60 - 140 |
120.000 - 23.000.000 |
||||
Name
medium
|
Matrix
|
Particle
size (mm)
|
Fractionation range (Da) |
|
|||
Superose
|
Cross-linked agarose |
|
|
|
|||
Superose 12 prep grade |
|
20 - 40 |
1.000 - 300.000 |
|
|||
Superose 12 |
|
8 - 12 |
1.000 - 300.000 |
|
|||
Superose 6 prep grade |
|
20 - 40 |
5.000 - 5.000.000 |
|
|||
Superose 6 |
|
8 - 12 |
5.000 - 5.000.000 |
|
|||
|
|
|
|
|
|||
Superdex
|
Cross-linked agarose linked to dextran |
|
|
|
|||
Superdex 30 prep grade |
|
22 - 44 |
-
10.000 |
|
|||
Superdex 75 prep grade |
|
22 - 44 |
500 -
30.000 |
|
|||
Superdex 75 |
|
11 - 15 |
500 -
30.000 |
|
|||
Superdex 200 prep grade |
|
22 - 44 |
1.000 - 100.000 |
|
|||
Superdex 200 |
|
11 - 15 |
1.000 - 100.000 |
|
|||
Applications
Preparative: purification of
proteins
-
Resolving power of the matrix (fractionation range)
-
Small sample volumes (< 5% of Vt)
-
Diffusion à broadening of
bands
-
Resolution is limited
Desalting of proteins
-
Group separation: proteins are separated from salt molecules
-
Small pore sizes: Sephadex G-25, BioGel P-6
-
Sample volumes up to 25 - 30% of Vt)
Applications
Analytical:
determination of molecular weight; pure proteins
Marker proteins
Influence of size and shape of
protein; hydrodynamic volume
Plot of Kav (or Ve)
versus log M
-
determination of oligomeric structure (subunit composition)
-
identification of prosthetic groups
-
gelfiltration
under denaturating conditions (in the
presence of unfolding reagents
-
Trouble-shooting
Poor resolution
Flow rate
(should be low)
Fractionation
range (narrow preferred)
Bead size
(small)
Sample size
(small, < 5% of column volume)
Low flow rate
Precipitates
on column à clean by washing with denaturating
agent (depending on matrix)
Skewed peaks
Adsorption of
proteins to column (elution after Vt) à
add salt
Air bubbles in
column à repack; de-aerate column material +
buffer
Different
polymerization states of a protein
Disappearance of protein activity
Adsorption of
protein to column à add salt
Separation of
subunits; loss of cofactors
Very common intermediate steps in a purification (link between subsequent purification steps)
May cause loss
of enzyme
Examples
Gelfiltration:
small sample volumes -> often concentration required; salt concentration is
not important.
Ion-exchange chromatography: binding to column at low salt con
centration
-> often desalting required; volume of sample is not important.
Dilution
Dialysis
Gel filtration
(desalting column)
Ultrafiltration
Ultrafiltration
Precipitation
Adsorption- and ion-exchange chromatography
Lyophilization (‘freeze-drying’)
Dialysis.
Ultrafiltration.
Ion-exchange chromatography
Principle
Separation of biomolecules according to charge
Stationary phase (column material) carries ionizable groups fixed by
chemical bonding
Fixed charged goups have counterions of opposite charge
Counterions can be replaced by appropriately charged biomolecules (e.g. proteins); proteins are bound to
the column by electrostatic interaction
Proteins can be removed from the column by change in elution
conditions: change in pH; addition of salt
Materials
Matrix
agarose, dextran, acrylamide, same as for gel filtration
exclusion limit
Charged groups
Positively charged groups à anion exchangers:
-DEAE di-ethylaminoethyl
-QAE quaternary
aminoethyl
Negatively charged groups à cation exchangers:
-CM carboxymethyl
-SP sulfopropyl
Steps in ion exchange
chromatography
1. Equilibration
Equilibrate with desired type and
concentration of counterion and pH. Easy exchangeable counterions; low
concentration of ions.
2. Sample application and adsorption
Binding of proteins with proper charge
to column by exchange of counterions. Removal of non-bound substances by
washing with application buffer.
3. Elution
of bound proteins
Elution by buffer
change: 1) increase of ionic strength or 2) change of pH. Stepwise or gradient
elution. Fractionation of bound proteins
4. Cleaning
(regeneration) of the column
Removal of strongly bound compounds
(high salt, NaOH)
5. Re-equilibration
Binding to ion exchangers
Binding is of proteins is dependent on:
1. Charge of the ion-exchanger (dependent on
pH of buffer in relation to pKa of charged group, e.g. DEAE has pKa of 9 - 9.5)
2. Net charge of protein (dependent on pH of
buffer in relation to pI of protein).
-
strongest binding to anion exchangers: pH above pI of protein
-
strongest binding to cation exchangers: pH below pI of protein
3. Ionic strength of buffer
-
low ionic strength à binding
-
high ionic strength à elution
Practice
-
Column dimensions: broad columns for high flow rates
-
Bed capacity (mg protein/ml gel under defined conditions); pore size
-
Sample volume not important; proteins concentrate on top of column when
bound
-
pH and ionic strength of sample
are important. pH must also be
compatible with stability of protein
-
Washing with loading buffer after application
-
Elution with increasing salt concentration or with change in pH (if
compatible with stability of enzyme)
-
Stepwise elution vs. gradient elution
-
Elution profile containing:
-
absorbance (A280)
-
activity (U/ml)
-
salt concentration (conductivity)
- enzyme purification and
concentration of enzymes
Setup for ion exchange
chromatography
Resolution
Rs = 2 x (distance
between peaks)/(average peak width)
= 2 (VR2 - VR1) / (wb1 + wb2)
Contributing factors
-
Retention factor
k': k' = (Ve - Vt)
/ Vt
k' negative for gel
filtration (Ve < Vt); (highly) positive for ion
exchange chromatography (Ve > Vt)
-
Efficiency factor
N: N = 5.54 (VR1/W½)2
Efficiency is dependent on
experimental conditions: column materials, packing, flow rate, sample volume
for gel filtration, air bubbles in column
-
Selectivity a: a = (VR2 - Vt) / (VR1
- Vt) (Z VR2
/ VR1)
Selectivity is a measure for
the distance between two peaks (steepness of gradient in ion-exchange
chromatography; fractionation range in gelfiltration)
Other expression for Rs:
Rs = ¼ (a -1) / a . N½ . k (1+k)
Even on columns with a bad selectivity often reasonable separation can
be achieved by good practice (high efficiency)
Protein
quantitation
1.
Absorption at 280 nm
(semi-quantitative)
Absorption
due to tryptophan (tyrosine) residues
Advantage:
- fast
Disadvantages:
- disturbance
by other substances with absorption at 280 nm
- absorption
different for different proteins, due to variation in Trp content
Rule of thumb: A280 = 1 corresponds to 0.5 – 2 mg.ml-1
protein
2.
Colorimetric methods
a.
(Micro-)biuret assay: Complex of Cu2+ with peptide
bonds (-CO-NH-) under alkaline conditions à Cu+-protein complex,
absorbance at 570 nm
b.
Lowry assay: Transfer of electrons from bound copper ions and from aromatic side
chains to Folin reagent
c.
Bradford assay: Adsorption of Coomassie Brilliant Blue G250 to protein (mainly Arg,
but also His, Lys, Trp, Tyr and Phe sidechains)
A
standard should be used in all colorimetric assays
(usually different amounts of bovine serum albumin, BSA)
Assay |
Sensitivity |
Accuracy |
Interference |
Biuret |
0 – 1 mg |
Very high, independent on aminoacid composition |
Amino groups [e.g.
(NH4)2SO4] |
Lowry |
0 – 0.1 mg |
Partially dependent on aminoacid composition |
Acids, chelators (EDTA), reductants
(DTT, phenol), (NH4)2SO4 |
Bradford |
0 – 0.01 mg |
Dependent on aminoacid composition |
Detergents (SDS, Triton X100, soap) |
To remove
small interfering substances, the protein can be precipitated with 5%
trichloroacetic acid (+/- 0.015% Na-deoxycholate)
Bioaffinity
chromatography
Principle
-
Separation based on specific reversible interaction of proteins with ligands
-
Ligands are covalently attached to solid support (gel matrix)
-
Chromatography on a bioaffinity matrix retains proteins with
interaction to the column-bound ligands
-
Proteins bound to a bioaffinity column can be eluted in two ways:
1. Biospecific elution: inclusion of free
ligand in elution buffer which competes with column-bound ligand
2. Aspecific elution: change in pH, salt, etc.
which weakens interaction protein with column-bound substrate
Because of specificity of the interaction, bioaffinity chromatography
can result in very high purification in a single step (10 - 1000-fold )
Protein-ligand
interaction
Kd
E + L ßà EL
When ligand is bound to solid support:
K'd
E + LM ßà E-LM
Binding of ligand to column should not interfere to much with
interaction with protein:
10-4 M < K'd < 10-10 M
Amount of enzyme retained on column (E-LM) dependent on:
-
K'd (interaction is to weak if K'd > 10-4
M)
-
Concentration enzyme in extract [E]
-
Degree of substitution of matrix [LM]
Biospecific elution with competitive ligand I dependent on:
-
Concentration of competitive ligand [I]
-
Interaction of competitive ligand with E-LM: Ki
Ligands
Small (bio)molecules : substrates, inhibitors, cofactors
Large (bio)molecules: antibodies, receptors, proteins
How to make
your own bioaffinity matrix.
1. Activation of matrix.
2. Covalent
attachment of spacer (linker) group with reactive end group.
3. Attachment of ligand to spacer
Activation of matrix
A common
procedure for sugar-based matrices is activation with cyanogen bromide:
Example 1:
Isolation of
an NAD-dependent dehydrogenase from a crude extract on NAD-agarose
1. LOADING
Column: NAD-agarose
Sample: crude extract with NAD-dependent
dehydrogenases 2. WASHING
Wash with high-salt
buffer to remove all non-bound proteins from column.
Proteins with
strong interaction with NAD (Kd = 10-4 – 10-10 M)
remain bound to the column
3. ELUTION
Bio-specific
elution of bound proteins with buffer containing 1 – 10 mM NAD
Free NAD competes
with column bound NAD
(Non-specific
elution by salt, pH,
temp. change)
Example 2: Isolation of an NAD-dependent dehydrogenase
from a crude extract with dye-affinity chromatography
Many
polycyclic textile dyes are competitive inhibitors for NAD(P) dependent redox
enzymes --> they can be used as ligands for affinity purification.
Cheap
Much more stable than NAD or AMP
agarose (dyes are not substrates
Different dyes have different
specificities. Interaction cannot be predicted.
Example 3: Fusion proteins with an affinity tag
1. Insertion
of gene for protein to be purified in a vector containing coding sequence for
an ‘affinity tag’ (e.g.
chitin-binding (CBP) tag, glutathione S-transferase (GST) binding tag).
Insertion in correct reading frame results in fusion protein.
Amino
acid sequence recognized by rare-cutting endoprotease sequence can be inserted.
Strong
promoter ensures high expression.
CPB fusion-expression vector
2. Expression
in E. coli results in high amounts of
fusion protein; after chromatography on affinity matrix (glutathione-sepharose,
chitin beads) fusion protein is specifically retained.
3. After on-column splicing (endonuclease) mature
protein can be eluted in almost pure form.
4. Regeneration
of the column by removal of tag (wash with glutathione; guanidinium chloride.
An affinity tag is very helpful for purification of proteins
without enzymatic activity (e.g.
regulatory proteins)
Hydrophobic Interaction chromatography
- Proteins are separated by hydrophobic interaction on columns with hydrophobic groups attached (e.g. phenyl-, octyl groups)
- Hydrophobicity of amino acid sidechains
Tryptofan > Isoleucine, Phenylalanine > Tyrosine > Leucine > Valine > Methionine
- Most
hydrophobic sidechains are buried in interior of protein, but some (clusters
of) hydrophobic groups occur at surface of protein
- Surface
hydrophobic sidechains can interact with hydrophobic groups for example
attached to a column.
Hydrophobic interactions
Hydrophobic groups in a aqueous
environment tend
to cluster (e.g.: oil
drops in water). Driving force: entropy of water
Hydrophobic interaction is dependent on:
- Ionic strength
Anions:
PO43- > SO42->
CH3COO- > Cl-
> Br- > NO3- > ClO4- >
I- > SCN-
Cations:
NH4+ > Rb+ > K+ > Na+ > Cs+ > Li+ > Mg2+ > Ca2+ > Ba2+
- Temperature
Increasing temperature --> stronger hydrophobic interactions
- Polarity of solvent
Compounds which reduce polarity decrease hydrophobic interactions (ethylene glycol)
- Hydrophobicity:
n-octyl > phenyl > n-butyl
Phenyl
substituted columns (e.g. Phenyl
Sepharose) are usually a good choice
Sample (application)
Sample and column in high concentration of a salt that promotes binding (for example ammonium sulfate just below the concentration that starts to precipitate protein)
-Wash with high salt buffer
Elution of bound proteins
- Negative gradient of salting-out ions (from high to low concentration)
- When protein is still bound at end of salt gradient: positive gradient of polarity-reducing compound (e.g. 0 – 50% ethylene glycol)
Proteins are eluted according to hydrophobicity
- Transition metals (Ni, Co, Zn) are attached to column
- Proteins
with affinity to metal ions can be bound to the column and subsequently eluted
by change of conditions
Not many
naturally occurring proteins have affinity for metal ions.
Technique is mainly used to purify recombinant proteins provided with a His-tag.
Resins for IMAC
Application of IMAC for purification of ‘His-tagged’
proteins
1. Provide
a protein with a ‘tag’ of 6 histidine residues by mutagenesis of the gene (add
codons for 6 consecutive His residues to the gene).
… ATC GCG TAG …
… Ile Ala Stop
… ATC GCG CAC CAT CAC CAT
CAC CAT TAG …
… Ile Ala His His His His
His His Stop
The His-tag should not interfere with folding or activity
of the protein (C-terminal is often the best choice)
2. Produce
the protein
3. Purify
the protein in 1 step with IMAC on a nickel column
4. Elute
the bound protein with imidazol (specific elution) or with aspecific elution
(pH)
Column
chromatography
1. Gel filtration.
Long
columns. Proteins do not
bind to column. Small sample volumes: < 5% of column volume, (for desalting:
up to 30%). Salt not important (usually appr. 0.1 M added to prevent adherence)
2. Ion-exchange
chromatography.
Proteins
will bind to columns depending on their charges: negatively charged proteins à
anion exchange column; positively charged proteins à
cation exchange column. pH of buffer is important. Sample volume not important.
Sample application at low
salt concentration; elution with gradient of increasing salt conc. or with pH-gradient.
3. Hydrophobic
interaction chromatography.
Proteins
will bind to column depending on their hydrophobicity. pH of buffer and sample
volume are less important. Sample application at high salt concentration; elution with a
gradient of decreasing salt conc. or
with hydrophobic solvents (ethylene glycol)
4. Bio-affinity
chromatography.
Proteins
with sufficient affinity to ligand will bind (Kd 10-4 -
10-10 M). Sample application at appr. 0.5 M salt. Elution with free
ligand (most specific) or with salt, pH, temp. gradient.
Order of steps in enzyme purification
Usually a purification has several steps.
1) Preparation of crude extract (cell lysis; removal of cell
walls/membranes in case of soluble proteins)
2) (Optional: removal of nucleic acids, ribosomes with protamine sulfate)
3a) (NH4)2SO4 precipitation; protein
to be purified should remain just soluble