Molecular Biology


2010 Fall Semester

Total credits/hours per week 4/4   (This semester 2/2)


課本 Compiled By Dr. Yen Lee

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:

Let’s watch this movie:

Course Outline

Mendel’s Discoveries

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


Frederick Griffith’s Experiment


Hershey and Chase Experiment


DNA Structure:(Download Chime [aMfehTFg]


1 hr



作業 1hr 10%



Matthew Meselson and Frank W. Stahl



RNA Structure


The Central Dogma


Transcription and Translation




Genetic Codes

The Direction of Protein Synthesis




Equilibrium constant



Free energy



RNA Secondary Structure


Amino Acids and Protein Structure

1 hr



作業  作業 1hr 10%

全班同學分工合作,將  網頁改編為中文,並將file 寄至  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]繳,逾期無分)


X-ray and NMR


Protein Structure Prediction


Different Protein Functions Arise from Various Domain Combinations




Allosteric Regulation

DNA grooves


DNA Strands Can Separate and Reassociating


DNA OD Reading and Melting Temperature


DNA Linking Number

Chromosome Structure


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.

Protein/Enzyme purification


Protocol modified from


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 presentation1/4.

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 : introductionmaterials and methodsresultsdiscussionsand 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:



salt concentration

oxygen sensitivity


mechanical forces


Purity-check:   SDS-PAGE


How is purification measured ?

Determination of specific activity

Physical methods: SDS-PAGE;


Preparing extracts for purification

Sources   -   

animal tissues



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


Oxygen                   anaerobic conditions for

oxygen-labile enzymes

Proteolytic enzymes  add inhibitors: EDTA, benzamidine, etc.

Storage                   frozen, suspension, addition

                                of glycerol

Mechanical            avoid foaming



buffering capacity


DpKa /DT

Henderson-Hasselbalch equation:

pH = pKa + log (base / conjugated acid)


Tris buffer:     Tris      +   HCl            pKa    8.0

Acetate buffer:    NaAc   +   HAc      pKa     5.0

Phosphate buffer: K2HPO4   +   KH2PO4      pKa     7.0


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




1.  Ammonium sulfate precipitation

2.  Gel filtration

3.  Ion-exchange chromatography

4.  Bio-affinity chromatography

5.     Hydrophobic interaction chromatography…..

Enzyme activity              

stoichiometry of participating reagents

requirement for cofactors


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 min-1 = 0.096

à Amount of enzyme in ADH stock solution:

     0.096    x 1000/10    x    50       = 480

                (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 (



3.               The activity of the enzyme solution (


Total amount of enzyme (U):

     Activity ( x volume (ml)


Specific activity (

     Activity ( / protein content (    


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:


pH (dependent on pI)


lipophylic agents (e.g. ethanol)

cross-linking agents (e.g. protamine sulfate)

water-extracting agents (e.g. polyethylene glycol)


Different proteins precipitate under different conditions


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



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)




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)





-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)




-                    Cleared samples

-                    Small sample volumes (< 5% of column volume)



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 size

of hydrated

beads (mm)

Fractionation range

for globular

proteins (Da)







40 - 120

               -              700

G-25 medium


50 - 150

      100   -           5.000

G-50 medium


50 - 150

      500   -         10.000



40 - 120

   1.000   -         50.000



40 - 120

   1.000   -       100.000



40 - 120

   1.000   -       150.000



40 - 120

   1.000   -       200.000





Sepharose CL

Cross-linked agarose





45 - 165

 10.000   -    4.000.000



45 - 165

 60.000   -  20.000.000



60 - 200

 70.000   -  40.000.000





Sephacryl HR

Cross-linked allyldextran/






25 - 75

  1.000   -        100.000



25 - 75

  5.000   -        250.000



25 - 75

10.000   -     1.500.000



25 - 75

20.000   -     8.000.000





Bio-Gel P






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




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











60 - 140

25.000   -    2.400.000



60 - 140

55.000   -    9.000.000



60 - 140

120.000 -  23.000.000

Name medium



size (mm)


range (Da)



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








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




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)



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




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





Desalting and concentration 

Very common intermediate steps in a purification (link between subsequent purification steps)


May cause loss of enzyme


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.

Desalting and buffer exchange



Gel filtration (desalting column)



Concentration of proteins



Adsorption- and ion-exchange chromatography

Lyophilization (‘freeze-drying’)








Ion-exchange chromatography



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



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




-                    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



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


-    fast


-       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 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)







0 – 1 mg

Very high,

independent on aminoacid


Amino groups

[e.g. (NH4)2SO4]


0 – 0.1 mg


dependent on



Acids, chelators (EDTA), reductants (DTT, phenol), (NH4)2SO4


0 – 0.01 mg

Dependent on




(SDS, Triton X100,


To remove small interfering substances, the protein can be precipitated with 5% trichloroacetic acid (+/- 0.015% Na-deoxycholate)


Bioaffinity chromatography 




-                    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 



E   +   L   ßà        EL


When ligand is bound to solid support:



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




Small (bio)molecules : substrates, inhibitors, cofactors


Large (bio)molecules: antibodies, receptors, proteins



文字方塊: Ligand				Specificity                                                   .
NAD, NADP			Dehydrogenases
5’-AMP				NAD-dependent enzymes
2',5'-ADP				NADP+-dependent enzymes
Glutathione	Glutathione-S-transferase (fusion proteins)
Chitin				Chitin binding protein (fusion proteins)
Amylose				Maltose binding protein (fusion proteins)
Blue B				Kinases, dehydrogenases
Blue F3G-A			NAD+-dependent enzymes
Red HE-3B			NADP+-dependent enzymes
Green A				CoA proteins, HSA, dehydrogenases
Lysine				rRNA, ds-DNA, plasminogen
Arginine				Fibronectin, prothrombin
Benzamidine			Serine proteases
Calmodulin				Kinases
Gelatin				Fibronectin
Polymyxin				Endotoxins
Heparin				Lipoproteins, DNA, RNA
Lectins, concanavalin A	Polysaccharides, glycosylated proteins
Antibodies (IgG)			Antigen (protein, etc)
Protein A				Fc antibody fragments
Protein G				Antibodies
Poly U, oligo-dT			Poly(A)+ mRNA

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




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



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.





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)



Surface hydrophobicity


-      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  


PO43- > SO42-> CH3COO- > Cl- > Br- > NO3- > ClO4- > I- > SCN-


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


Immobilized metal affinity chromatography




-       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



 … 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