MIC/BIO/BCH522

Spring 2006

Protein Purificationor

“How not to waste good clean thoughts on dirty enzymes”A. Kornberg

Scheme

1. Source of the protein2. Lysis Methods3. Ammonium Sulfate fractionation4. Dialysis5. Gel Filtration 6. Ion exchange chromatography7. Protein storage 8. Assays for activity9. Assays for contamination

– Nuclease– Nucleic Acid– Nucleoside triphosphates

Initial issues to consider……1. What type of protein will be expressed?

- prokaryotic origin, the obvious choice is to use E. coli. - if the protein is from a eukaryotic source, the method of choice will depend on protein solubility, post-translational modification and codon usage

2. Is the protein expressed in E. coli soluble?- many eukaryotic proteins don't fold properly in E. coli and form insoluble aggregates (inclusion bodies). - is possible to resolubilize the protein from the inclusion bodies or express at a lower temperature. - a fusion protein with a highly soluble partner such as glutathione-S-transferase (GST), maltose binding protein (MBP)

3. Does the protein require post-translational modifications for structure/activity?- complex modifications, like N- and O-glycosylation, phosphorylation, are carried out exclusively by eukaryotic cells.

4. What is the codon usage in the protein?- Not all of the 61 mRNA codons are used equally- major codons in highly expressed genes; minor or rare codons tend to be in genes expressed at a

low level. - Which of the 61 codons are the rare ones depends strongly on the organism. - The codon usage per organism can be found in the Codon Usage Database- Usually, the frequency of the codon usage reflects the abundance of their cognate tRNAs. - The following problems are often encountered:

Interrupted translation (truncations); Frame shifting; Misincorporation of amino acids

Expression system1. Escherichia coli

– The expression of proteins in E. coli is the easiest, quickest and cheapest method. – There are many commercial and non-commercial expression vectors available with different

N- and C-terminal tags and many different strains which are optimized for special applications

– Vectors which do not add tags are also available with strong promoters

2. Eukaryotic cells– Human cells– Xenopus– Baculovirus infected insect cells

• Insect cells are a higher eukaryotic system than yeast and are able to carry out more complex post-translational modifications than yeast or E.coli.

• They also have the best machinery for the folding of mammalian proteins and, therefore, give you the best chance of obtaining soluble protein when you want to express a protein of mammalian origin.

• The disadvantages of insect cells are the higher costs and the longer duration before you get protein (usually 2 weeks).

– Yeast• has some advantages and disadvantages over E. coli. • One of the major advantages is that yeast cultures can be grown to very high

densities, which makes them especially useful for the production of isotope labeled protein for NMR.

• The two most used yeast strains are Saccharomyces cerevisiae and the methylotrophicyeast Pichia pastoris.

Comparison of expression systems

Characteristics E. coli Yeast Insect cells Mammalian cells

Cell growth rapid (30 min) rapid (90 min) slow (18-24 h) slow (24 h)

Complexity of growth medium minimum minimum complex complex

Cost of growth medium low low high high

Expression level high low - high low - high low - moderate

Extracellularexpression

secretion to periplasm secretion to medium secretion to medium secretion to medium

Posttranslational modifications

Protein foldingrefolding can be

requiredrefolding may be

required proper folding proper folding

N-linked glycosylation none high mannose simple, no sialic acid complex

O-linked glycosylation no yes yes yes

Phosphorylation no yes yes yes

Acetylation no yes yes yes

Acylation no yes yes yes

gamma-Carboxylation no no no yes

Vectors to use in E. coli

Promoter source regulation induction Expression level

lac E. coli lacI, lacIq IPTG low

tac (hybrid) E. coli lacI, lacIq IPTG moderately high

trc (hybrid) E. coli lacI, lacIq IPTG moderately high

araBAD E. coli araC l-arabinose variable

pL λ l cI857 (ts) shift to 42°C moderately high

T7-lac operator T7 lacIq IPTG very high

Cell growth and expression• Often, the plasmid must be maintained in a cloning strain and transformed into the expression strain just before use• Following transformation, several individual colonies should be picked and expression verified using SDS-PAGE• The most promising isolate should be used to generate a starter culture. This culture can be used to inoculate a

larger culture in which expression will be induced• The starter culture can be grown in the presence of 0.2 – 0.4% glucose if the promoter is IPTG induced• Typically, induction cultures will contain one or more antibiotics• The use of ampicillin requires special care. The selectable marker, β-lactamase, is secreted into the medium where it

hydrolyzes all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture

• Some possible solutions are:– grow overnight cultures at 30°C or lower. – spin overnight cultures and resuspend the pellet in fresh medium to remove β-lactamase. – use the more stable carbenicillin instead of ampicillin– spike the cultures with ampicillin at the point of induction.

• The induction culture must be aerated as well as possible – this is CRITICAL!! For good aeration, don't use more medium than 20% of the total flask volume.

• Inoculation of the main culture and incubation until OD600 reaches 0.4-1. The optimal OD value depends on the culture method and the medium. For flask cultures using LB-medium an OD600 of 0.6 is recommended. To increase the growth rate, we carry out the cultures at 37°C until the OD for induction is reached.

0 2 4 6 8 10 12 140.1

1

10

OD600 = 0.4 to 0.6

IPTG

Time (hours)

OD

600

Protein purification table

StepOD260 (x dil)

OD280 (xdil)

Total protein (mg)

Total activity (units)

Specific activity

(units/mg) Purification % Yield

Crude cell lysate 1200 550 5500 6600 1.2 NA NA

30-70% Am. Sulf. cut 204 102 1020 5910 5.8 4.8 89.5

DEAE Sephadex 37 18.7 187 5070 27.1 4.7 85.8

CM Sephadex 6 10.2 102 4420 43.3 1.6 87.2

Phenyl Sepharose 3.8 5.6 56 3930 70.2 1.6 88.9

Gel Filtration 1.8 3.2 32 2970 92.8 1.3 75.6

Affinity resin type #1 0.25 0.58 5.8 2520 434.5 4.7 84.8

Affinity resin type #2 0.27 0.53 5.3 2390 450.9 1 94.8

Good record keeping is absolutely essential - monitor protein by SDS-PAGE and OD280– the amount of protein, the level of contamination and where possible,

Specific activity should be determined at each stage

Lysis methods for E. coliPhysical methods:• Sonication.

– the most popular technique for lysing small quantities of cells (1-6 L of cell culture). – Cells are lysed by liquid shear and cavitation. – DNA is also sheared during sonication, – The main problem is controlling the temperature addressed by:

• keeping the suspension on ice • using a number of short pulses (5-10 sec) with pauses (10-30 sec) to re-establish a low temperature.

• Homogenization.– Homogenizers are the most common devices to lyse bacteria. – cells are lysed by pressurizing the cell suspension and suddenly releasing the pressure. This creates a liquid

shear capable of lysing cells. – the French press ; uses 6000-10,000 psi. Multiple (2-3) passes are generally required to achieve adequate lysis.

• Freezing and grinding.– An alternative lysis method is to freeze the cells directly in liquid nitrogen and ground the frozen cells to a

powder using a mortar and pestle that are chilled with liquid nitrogen. The powder can be stored indefinitely at -80°C and the cell lysate can be prepared by adding the powder to 5 volumes of buffer.

Enzymatic and detergent lysis:• Enzymatic lysis

– based on the digestion of the peptidoglycan layer of the bacterial cell wall by lysosyme. – Gram-negative bacteria, however, have an outer membrane that is external to the cell wall and needs to be

permeabilized to expose the peptidoglycan layer. – Tris, often used as a buffer in lysis methods, effectively permeabilizes outer membranes. – Solubilization is enhanced by the addition of EDTA (1 mM).

• Detergents Non ionic or zwitterioninc

sodium deoxycholate, brij-35, CHAPS, Triton x-100

DNA removal• Most lysis methods cause the release of nucleic acids – both DNA and RNA • These have to be removed because they can cause viscosity problems or they can interfere with

subsequent chromatographic steps. • Different methods exists: • Enzymatic digestion by the addition of DNase I (1 µg/ml) to the cell lysate. The mixture is

incubated on ice for 10-15 min. • Mechanical breakdown by shearing during sonication. • When the French Pressure Cell is used it is advisable to add DNase I to the cell suspension. • Nucleic acids can sometimes be readily removed from the sample by the addition of large

cationic compounds such as polyethyleneimine, or streptomycin sulfate. • The nucleic acids bind to these compounds via electrostatic interactions and the complex

precipitates and can be removed via centrifugation.• Add the precipitants to the cell lysate and incubate the solution for 30 min at 4°C. • Precipitation by treatment with polyethyleneimine (0.1% (w/v)) or protamine sulphate (1%

(w/v)) followed by centrifugation. • Often, the protein of interest will also be precipitated using these cationic agents and therefore

must be extracted from the resulting pellet

Protein precipitation methods• Protein stability in solution dependent on:

– electrostatic interactions (ionogenic, amino acid side chains; salts)– H-bridges (side chains; 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)

• Different proteins precipitate under different conditionsFRACTIONATION

• Polyethylene glycol– neutral, non-denaturating compound– low heat of solution steric exclusion mechanism (binds water)

• Protamine sulfate– small, basic proteins from sperm (large #’s of Arg and Lys residues)– precipitation of large protein complexes (ribosomes), DNA, RNA by complexation– precipitation is concentration dependent

• Apolar solvents (acetone, alcohol) and Trichloroacetic acid– unfolds/denaturates proteins

• Ammonium sulfate

Why ammonium sulfate ?• Most proteins precipitate in saturated solution (4 M)• Low heat of solubilization ( prevents denaturation of proteins)• Low density of saturated solutions (1.25 g/cm3) proteins can be collected in pellets by

centrifugation• Concentrated solutions prevent microbial growth• Protects most proteins from denaturation• Cheap

• Applications of 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: specific amounts to be added to reach a saturation level– from a saturated (= 100%, w/v) solution

• Note: saturation level is temperature dependent !!

Ammonium sulfate precipitation theory• The solubility of protein depends on, among other things, the salt concentration in the solution. • At low concentrations, the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting

protein into the solution and enhancing the solubility of protein. This is commonly known as salting-in. • as the salt concentration is increased, a point of maximum protein solubility is usually reached. Further increase in the

salt concentration implies that there is less and less water available to solubilize protein. Finally, protein starts to precipitate when there are not sufficient water molecules to interact with protein molecules. This phenomenon of protein precipitation in the presence of excess salt is known as salting-out.

• Many types of salts have been employed to effect protein separation and purification through salting-out. • Of these salts, ammonium sulfate has been the most widely used chemical because it has high solubility and is relatively

inexpensive. • There are two major salting-out procedures:

– In the first procedure, either a saturated salt solution or powdered salt crystals are slowly added to the protein mixture to bring up the salt concentration of the mixture.

– The precipitated protein is collected and categorized according to the concentration of the salt solution at which it is formed. This partial collection of the separated product is called fractionation.

– The protein fractions collected during the earlier stages of salt addition are less soluble in the salt solution than the fractions collected later.

• Whereas the first method just described uses increasing salt concentrations, the following alternative method uses decreasing salt concentrations.

• In this alternative method, as much protein as possible is first precipitated with a concentrated salt solution. Then a series of cold (near 0ºC) ammonium sulfate solutions of decreasing concentrations are employed to extract selectively the protein components that are the most soluble at higher ammonium sulfate concentrations. The extracted protein is recrystallized and thus recovered by gradually warming the cold solution to room temperature. This method has the added advantages that the extraction media may be buffered or stabilizing agents be added to retain the maximum enzyme activity. The efficiency of recovery typically ranges from 30 to 90%, depending on the protein. The recrystallization of protein upon transferring the extract to room temperature may occur immediately or may sometimes take many hours. Nevertheless, very rarely does recrystallization fail to occur. The presence of fine crystals in a solution can be visually detected from the turbidity.

Dialysis• After an ammonium sulfate precipitation step, or an ion exchange chromatography step, the protein of interest may be

in a high salt buffer; salt may hinder the next purification step. • One of the most common methods to remove salt is that of dialysis• The method of dialysis makes use of semi-permeable membranes. • The main feature of this membrane is that it is porous - the pore size is such that small salt ions can freely pass

through the membrane, larger protein molecules cannot (i.e. they are retained). Thus, dialysis membranes are characterized by the molecular mass of the smallest typical globular protein which it will retain.

• This is commonly referred to as the cutoff of the tubing (e.g. Spectrapore #6 dialysis tubing has a cutoff of 1,000 Daltons, meaning that a 1,000 Dalton protein will be retained by the tubing but that smaller molecular mass solutes will pass through the tubing)

• Dialysis proceeds by placing a high salt sample in dialysis tubing (i.e. the dialysis "bag") and putting it into the desired low salt buffer:

• One consequence of dialysis to watch out for is that while salt ions are moving out of the bag, water molecules are moving into the bag – thus the bag will swell (and protein concentration will decrease)

• In the extreme case, the bag may actually swell to the point of rupture. • it is a good idea not to fill the bag completely, but leave a void/space to allow for potential swelling.

For how long and against what volume do you dialyze samples?

• A useful rule of thumb is that for most types of dialysis tubing the dialysis is 80% complete after four hours• At equilibrium the salt concentration of the sample can be calculated as follows:

• The buffer volume for dialysis is a function of the required final concentration of salt in the sample• Dialysis example

– a 10ml protein sample from an ion exchange column elution pool which contains 1.0M NaCl. – the next step in the purification requires no more than 1mM NaCl in the sample.

– Note that in the above example this would commonly be referred to as a "1:1,000" dialysis. – However, the same results can be achieved with two sequential "1:32" dialyses (i.e. the square root of the

1:1,000 dialysis - in other words, two sequential 1:32 dialyses is equivalent to a single 1:1,000 dialysis): – First dialysis versus 310 ml of buffer: sample NaCl conc will be (10*1.0)/(320) = 31 mM– Second dialysis versus 310 ml of buffer: sample NaCl conc will be (10*0.031)/(320) = 0.97 mM

– Thus, instead of making 10 L of buffer, we could make only 620 ml and achieve the same results with two dialysis steps

Concentration of samples• Frequently, a particular step will dilute the protein sample• One simple method to concentrate samples is to use a semi-permeable membrane (dialysis bag)• Here, the sample in a dialysis bag is coated with a high molecular weight solute which can readily

be dissolved by the buffer. – - polyethylene glycols and polyvinyl pyrolidones, MW = 20,000 Da.

• If our sample in a dialysis bag is coated with dry forms of the above polymers, water will be removed from the dialysis bag and hydrate the polymer on the exterior

• The result is a decrease in volume of buffer in the dialysis bag,• i.e., the protein becomes concentrated

• Following concentration using a dialysis bag, the bag has to be rinsed with dH20 to remove excess polymer and re-dialyzed to ensure the buffer is equilibrated to the next purification step

• Protein can be concentrated in a variety of other ways– Amicon spin columns– Ammonium sulfate precipitation (if the concentration is not too low)– Certain ion exchange columns can achieve significant concentration, i.e., MonoQ

Chromatography• Usually several different chromatographic steps are performed. • There is no set order, but a typical order might be cation exchange, gel filtration, and then anion

exchange. • i) Gel filtration - separation on the basis of size

– The matrix contains pores that allow smaller molecules to enter but excludes larger molecules. – larger molecules spend less time in the matrix and elute first. – The size of the protein can be estimated from the elution time or volume.

• ii) Affinity– resin contains a specific group (i.e. ligand or antibody) that causes the protein of interest to bind – ligand affinity, elution can be accomplished by the addition of excess ligand. – antibody based affinity columns the protein-antibody binding must be weakened by changes of the

pH and/or the salt concentration.• iii) Ion exchange

– The protein sticks to these resins by electrostatic interactions. – Resins contain either negative (cation exchange resins) or positive (anion exchange resins)

charges. – In very general terms a protein will stick to a cation exchange resin below its pI and to an anion

exchange resin above its pI. – what really matters is the local charge distribution on the surface of the protein (i.e. patches of

residues with similar charges).– Elution of proteins from ion exchange resins involves either a change in pH that results in a change

in the charge of the protein or by increasing the salt concentration. – Increasing salt provides additional ions than compete with the protein for binding sites on the resin.

Gel filtration• Gel filtration does not rely on any chemical interaction with

the protein, rather it is based on a physical property of the protein - that being the effective molecular radius (which relates to mass for most typical globular proteins).

• Gel filtration resin can be thought of as beads which contain pores of a defined size range.

• Large proteins which cannot enter these pores pass around the outside of the beads.

• Smaller proteins which can enter the pores of the beads have a longer, tortuous path before they exit the bead.

• Thus, a sample of proteins passing through a gel filtration column will separate based on molecular size: large ones will elute first and the smallest will elute last (and "middle" sized proteins will elute in the middle).

• There are two extremes in the separation profile of a gel filtration column.

• There is a critical molecular mass (large mass) which will be completely excluded from the gel filtration beads. All solutes in the sample which are equal to, or larger, than this critical size will behave identically: they will all eluted in the excluded volume of the column

• There is a critical molecular mass (small mass) which will be completely included within the pores of the gel filtration beads. All solutes in the sample which are equal to, or smaller, than this critical size will behave identically:they will all eluted in the included volume of the column

• Solutes between these two ranges of molecular mass will elute between the excluded and included volumes

Elution of proteins from a gel filtration column

• If the total column volume is Vt and Vx is the volume occupied by the resin, then the volume surrounding the resin is Vo:

Vt = Vx + Vo

• The elution volume, Ve, is the volume of solvent required to elute the protein. The molecular weight of the protein can be obtained from the following formula:

Ve/Vo = a*log(Mr) + b• Where "a" and "b" are constants obtained by

calibration of the gel filtration column.• Note that the native molecular weight is obtained,

e.g. hemoglobin would give a measured molecular weight of 62 kDa and myoglobin (Mr = 17 kDa) would elute later.

Protein Native Mr (Da) #SubunitsMyoglobin 17,200 1TIM (triosephosphate isomerase) 53,300 2Hemoglobin 62,000 4IgG (immunoglobulin G) 140,000 4ATCase (aspartate transcarbamoylase) 307,900 12

Ion exchange• Ion exchange resins contain charged groups. • These may be acidic in nature (in which case the resin is a cation exchanger) • or basic (in which case it is an anion exchanger). • Cation and anion exchangers may be broken down further into weak and strong exchangers (reflecting binding

affinity). • Usually, samples are loaded under low salt conditions and bound material is eluted using increasing salt

concentration. • Proteins bound to ion exchange resins are bound via non-covalent ionic (salt-bridge) interactions. • Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric

point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI. • There are two general types of methods when eluting with a salt solution:

– Gradient elution– Step elution

• A gradient elution refers to a smooth transition of salt concentration (from low to high) in the elution buffer. Weakly binding proteins elute first, and stronger binding proteins elute last (i.e. they require higher salt concentrations in the buffer to compete them off the column)

• A gradient salt concentration can be made using a gradient maker.

Elution profiles

Linear gradient Step-wise elution

•If we know the concentration range of salt over which a protein of interest will elute we can simply elute with a buffer containing that concentration of salt. This is known as a step elution. •Step elutions are generally faster to run, and elute the protein in a smaller overall volume than with gradient elution. •They generally work best when contaminants elute at a significantly different salt concentration than the protein of interest

A typical ion exchange chromatogram

Linear gradient

Resolving peaks and pooling for purityPanel A:• Contaminating peaks will not necessarily be

completely separated from the peak which contains our protein of interest

• In the following picture there are two components being resolved, and they are present in equimolaramounts (thus, the starting purity is 50%). The yield and purity are listed for the situation where we were to pool each peak by splitting at the midpoint between them (in this particular example the yield and purity are identical in each case)

• This gives you some idea of the amount of cross-contamination in each peak as a function of their separation from one another.

Panel B• These are all the same chromatogram, however, we

can pool them differently to get better purity (at the expense of yield

• The blue peak is the peak of interest and it is not resolved from a contaminating peak (in red).

• The vertical line represents the left-most fraction we use to pool the peak (we pool all fractions to the right of the vertical line to get our protein of interest)

• In the last panel we see that we can achieve about 98.8% purity if we are willing to part with half our protein!

A. B.

Storage of proteins• After you have invested all the hard work to express and purify your target protein, you should not forget to think

about how you want to store your purified protein. • The method of choice depends completely on the stability of the protein and on when and how you want to use it

later on. • For short term storage (up to 24 h), most proteins can be kept at 4°C.• For long term storage (more than a week), it becomes necessary to freeze the protein preparation • Protein preparations will be stable for several years at -80°C or even in liquid nitrogen. • For some proteins, it is important to freeze it rapidly using liquid nitrogen or a dry ice/ethanol mixture to avoid

denaturation. • It is also important to freeze the solution in small aliquots to avoid repeated freezing and thawing which may

reduce the biological activity or affect the structure. • Several stabilizing agents can be added, such as glycerol (5-50% (w/v)), serum albumin (10 mg/ml), reducing

agents (such as 1 mM DTT), and salt (i.e., NaCl, KCl at 20-150 mM, depending on the protein)• To transfer the protein preparation into the storage buffer:

– Add an equal volume of pure glycerol to the protein preparation.– Dialyze the protein preparation against the storage buffer containing 50% glycerol. This method has the

additional advantage that it results in an approx. threefold concentration. •

Alternative methods are:– Storage of the protein at 4°C as an ammonium sulfate suspension. – Storage of the protein at 4°C or lower in a lyophilized form. – For the lyophilization it is necessary that the protein is dissolved in a volatile buffer (such as

trimethylamine/HCl; pH range 6.8-8.8). – Note that not all proteins are stable during the freeze-drying process.

Concentration determination• Several methods exist,

– UV Absorbance (280 nm)– Bradford Assay– BCA (PIERCE), (modified Lowry)– Lowry

• Most protein assays utilizes an internal standard (e.g., BSA or lysozyme) to generate a reference or standard curve. The protein of choice is then measured against the standard curve. The protein used for the standard curve will affect the concentration of your purified protein sometimes by several orders of magnitude

• Optimal method is to use the extinction coefficient of the purified protein• Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent

on the amount of Phe and disulfide bonds). • Therefore the A 280 varies greatly between different proteins; for a 1 mg/mL solution, from 0 up to 4 for some

tyrosine-rich wool proteins, although most values are in the range 0.5-1.5 (Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Anal. Biochem. 68, 465-484).

• The advantages of this method is that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined.

• The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.

• Can also provide insight into the degree of aggregation of the protein preparation (A 340 )

RuvA and SSB – two homotetramers

RuvA

250 280 310 340 370 4000.00

0.05

0.10

0.15

0.20

OD280 = 0.144OD260 = 0.088OD340 = 0.013

ε = 5.55 x 103 M-1 cm-1

Wavelength (nm)

Abs

orba

nce

(a.u

.)

SSB protein

250 280 310 340 370 4000.0

0.5

1.0

OD280 = 0.755 OD260 = 0.41OD280 = 0.510 OD260 = 0.26OD280 = 0.180 OD260 = 0.11

ε = 3 x 104 M-1 cm-1

Wavelength (nm)

Abs

orba

nce

(a.u

.)

Final gel from an EcoR124I preparation

0 100 2000

25

50

75

100

S(1.0)

M(2.4)

R(2.0)

distance migrated (mm)Re

altiv

eIn

tens

ity(a

.u.)

M 1 2 3 4

R-subuinit

M-subunit

S-subunit

A B

209

12480

49

35

A. Lane 1 = cell lysate; lane 2 = ammonium sulfate pellet; lane 3 = pooled fractions following Q-sepharose chromatography; lane 4 = final pool (pooled fractions following Heparin FF chromatography and overnight dialysis

against storage buffer) B. Quantitation of lane 4 band intensities. The peaks labeled R, M and S, correspond to the R-, M-

and S-subunits respectively. The numbers in parentheses indicate the relative area under each peak.

Assays for activity

• Since these are DNA binding proteins that we are attempting to isolate, all activities should be DNA-dependent

• And the proteins should bind DNA (or RNA)• DNA binding assays using gels, filter binding or

fluorescence based assays• ATPase assays• These assays provide information both on the

presence of activity, DNA-dependence of activity and also provide the fraction of active protein in an assay

SSB site size

0 10 20 300

25

50

75

100

125

1μM SSB

2μM SSB

8μM nts 14 μM nts

M13 ssDNA μM nts

Frac

tiona

l flu

ores

cenc

e (%

)

Stoichiometry for EcoR124I

0 10 20 300

10

20

30

40

50

60

12.4 nM/2nM DNA= 6.2nM or 16% active

[protein] (nM)

Rat

e ( μ

M/m

in)

0 10 20 30 400.0

0.5

1.0

1.5

2.0

scDNA

linear

no DNA

M13 RF

Time (min)

Abs

orba

nce

at34

0nm

(a.u

.)

Phase I; 34 M/minμ

Phase II; 2.4 M/minμ

23 M/min μ

DNA-dependent and

site specific ATPase activity of EcoR124I

Sources of contamination

• Nucleases Nucleases Nucleases!!!• E. coli has 12 exonucleases, several endonucleases, phosphatases, ribonucleases• It is imperative that a variety of nucleases asays be done following purification to determine

whether any nucleases are present• These assays typically involve incubating the purified protein with radioactively labeled DNA• Subject the samples to electrophoresis and compare to control untreated DNA• Types of DNA used (in separate assays):

– Linear dsDNA– ssDNA (M13 for example)– Linear ssDNA (either oligonucleotides or linearized M13 mp8 ssDNA)

• If nucleases with distinct polarities are anticipated, both 5’- and 3’-end labeled substrates should be used

• Nucleic acid – either DNA or RNA– can be detected using spectrophotometric methods and via radioactive labeling