Protein Purification Lecture (Ostap)

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September 13, 2010

Michael OstapOffice: B40 Anat-ChemEmail: [email protected]: 215-573-9758

Methods for the mani ulation of roteins:

• Expression

• Purification

• Detection/Quality control

• Inventory of the participating molecules

• Atomic structure of key molecules.

• Identification of the molecular partners of each component in the system.

• Rates of reactions and affinities of partners for each other.

• Tests for physiological function for each molecule and its role in disease.• A mathematical model for the whole system.

** See review by Pollard that I put on Blackboard.

Don't waste clean thoughts on a dirty enzyme.

Efraim Racker

** See review by Kornberg that I put on Blackboard.



- Nearly all lecturers will present experiments that utilize purified proteins.

- Almost all of you are going to need to purify a protein at some point.

ey quest ons:

1) Is there a cheap, abundant source of the protein?

• What organism?• Which tissue?• Which subcellular compartment?• ow muc pro e n w e nee e

2) Is the protein easy to purify from natural sources?

• What is the relative abundance of the target protein?• How stable is the protein?• Is there an appropriate purification scheme?

Key questions:

3) Is the protein soluble or membrane-bound?

4) Is solubility in aqueous buffers important?

5) Will there be a need for:

•site directed mutagenesis?•domain dissection?•chimeras?

When should a native source for the protein be used:

1. Gene not available (unlikely).

2. Protein is naturally abundant and easy to purify.(Actin, Myosin, Microtubules, Hemoglobin, serum albumin…)

3. Expression of recombinant protein in heterologoussystems is problematic.(Multisubunit complexes – becoming less of a problem)

When should a recombinant protein be used:

1. Native protein is present in low abundance.

. rote n ar to pur y rom nat ve sources.

3. Removal of non-essential features desirable.

4. Genetic analysis desirable.



Recombinant protein production

1. Obtain a cDNA clone of your protein of interest

2. Decide on an expression system and purification scheme.Clone the cDNA into a vector with the appropriate promoter.

3. Optimize expression

. ur y e pro e n

5. Protein characterization and quality control

Recombinant protein productionSte 1: Obtainin a DNA clone

1. “Cloning by internet”: clone is obtained from

another lab or purchased from a bank or a company.

$$


2. PCR cloning: A pair of PCR primers are designed to match

the regions of the target gene encoding the N- and C-terminiof the protein to be made. The obtained PCR product is thensubcloned into an appropriate plasmid.

PCR Sequence&PCR/RT-PCR product subclone

cDNA library/ polyA mRNA

Note:Note: Full-length proteins or truncates

can be generated by this method.


3. Homology cloning: The target clone is identified and

isolated from a cDNA library via screening of the library(harbored in a plasmid/phage-bearing bacterial strain) witha hybridizing DNA/antibody probe.

Screen w/probe Isolate clone &

recover plasmid

Replica filter

Sequence-check

plasmid insert

Master plate w/colonies



Recombinant protein productionSte 2: Subclonin for overex ression

1. Choice of organism: time-money-amount-quality

Prokaryotic systems: Fast, cheap, high throughput

- sc er c a co : os popu ar, es or cy op asm cproteins. Variety of overexpression vectors.

Eukar otic s stems: Ex ensive laborious hi h fidelit

- Yeast: Simplest system with secretory pathway.Will glycosylate proteins. Disulfides will form.

- nsect ce s: ecretory pat way. ore e a orate g ycosy at onpatterns. Disulfides will form. High levels of expression.- Mammalian cells: High fidelity for postranslationalmodifications and maturation. Ver ex ensive.

Eukaryotic systems provide native post-translational processing.

Expression System Insect Cells E. Coli Yeast Cells Mammalian

Proteolytic Cleavage +/- +/- +/- +Gl cos lation -Secretion + +/- + +

+ - + - + - +Phosphorylation + - + +

cy a on

+ - + +Amidation + - - +Percent Yield 1-30% 1–5% 1% < 1%

Recombinant protein productionSte 2: Subclonin for overex ression

2. Choice of vectors: Once the choice of organism is made,

Promoter: Constitutive or inducible? Strong or weak?

Selectable marker: non-overla in with other lasmids.

Purification/detection tags: affinity tags, epitopes, GFP.

Folding/solubility/secretion tags: thioredoxin, GST, protein A

Tag removal features: restriction protease sites, inteins.

Insert is pasted into multiple cloning site of vector by using compatiblerestriction endonuclease ends, or by DNA recombination.

The pET system: a phage T7 RNA polymerase-driveninducible s stem ver o ular for ex ression in E. coli)

Inducer: IPTG (Isopropyl thiogalactoside), a lactose analogue

BL-21 DE3

Note: In pRSET (Invitrogen) the lac operator is not present, so the system is “leakier”.



Redox state influences the expression of insoluble proteins-.

-Role of thioredoxin in protein solubility not entirely understood.

-For some secreted proteins, mutant strains with altered cytoplasmic redoxbalance promote disulfide formation and correct folding (tied tosolubility).

Baculovirus-mediated insect cell expression-

-

yeast.

- Several other post-translational modifications available.

- Moderate to hi h ields.

- Insect cell lines

Trichoplusia ni (T.ni)Drosophila melanogaster (S2)

Virus-mediated protein overexpression in insect cells

- A recombinant baculovirus genome bearing the target gene is generated

in E. coli cells (“bacmid”) or in insect cells.

- Utilize late promoters (polyhedrin)

- Bacmid-transfected insect cells produce and release virus.

Virus-free insect cell systems allow for constitutivepro e n pro uc on



Drosophila melanogaster cells: alternate route to more.

Amplification factor given bymultiple plasmid copies

’

Mammalian expression vectors: pcDNA family

- Ideal in terms of post-translational modifications- , - .

- Low to moderate yields for most proteins.

Vectors for secreted proteins

- Signal peptide-containing vectorsallow for efficient RER insertion.

- Transmembrane domains allow for

protein retention at the cell surface.

Vectors including specialized signals allow for targetingo spec c n race u ar compar men s



Inducible systems for mammalian cell expression:A tetracycline repressor/VP16 activation domain (HSV)

hybrid system

Tet responsive

Mutated form

Tackling complexes: dual expression from one vector

RETROVIRAL

-Highest levels of transduction.

-Strong expression.

Recombinant protein productionSte 3: Protein over roduction

Once cells are transfected with the recombinant plasmid,

-What is the effect of time?

- re n ucers requ re , ra, e w a eve s

-Are repressors/silencers needed (Dox, Tet) (toxicity)

-Is the protein unstable? (temperature, protease- strains)

-Is the protein soluble? Does it matter?

-Does media formulation affect expression levels (coli )?

Parameters are adjusted empirically. 10-100 ml pilot runs

done, systematically varying conditions.



Recombinant protein productionSte 3: Protein over roduction

Scale-up: The size of a preparative run will depend on specificneeds, the expression/purification final yield per liter of culture andthe setup used for culturing.

- High yields (40-400 mg protein/liter) may be obtained usingaffinity-tagged proteins in high expression systems (pET,baculovirus)

- Fermentation setup results in higher yields because of improveddiffusion of oxygen and nutrients into the culture, allowing for much

.

Recombinant protein productionSte 4: Protein urification

contaminants as swiftly and efficiently as possible.

How much to purify?:“Functionally pure” vs “homogeneously pure”

Critical steps in any purification scheme:1. Capture2. Separation3. o s ng

1. Capture

The objective is to separate the target protein from the bulk medium orce ysa e o preven egra a on

- Inactivation of proteases (protease inhibitors)- Removal of nucleic acids (DNAse I, PEI)

- Affinity tags (polyHis, FlagTag, GST, MBP)

- Preci itation ste s saltin out: ammonium sulfate

- Addition of stabilizers (osmolytes, redox reagents)

relatively short times.

2. Separation

The objective is to remove most contaminants from the preparation. Time iscritical depending on the stability of the target protein.

-(anion/cation exchangers), dyes, affinity ligands, hydroxylapatite, HIC.

-> Design purification scheme so as to minimize sample preparationbetween steps (avoid dialysis).



ION EXCHANGE CHROMATOGRAPHY

- Proteins bind to a charged matrix by exchange with counter-ions.

-

the mobile phase in a step-wise or continuous fashion. A280

a t

AFFINITY CHROMATOGRAPHY

- A ligand (substrate, antibody, tag, co-factor) is immobilized to a matrix.

- Target protein binds selectively to matrix. Eluted with high ionic strengthor competitor molecule).

Metal chelate chromatography: A simple, powerfulaffinity principle for tagging

o y s ne a s en o s ac , us as ases o. s a ows em oform coordination complexes with divalent Nickel attached to amatrix through a chelating agent.

NTA: Nitrilotriacetic acid (chelating agent).



Maltose bindingprotein (MBP):

proteinpurified overamylose resin

3. Polishing

The objective in this step is to remove minor or persistent contaminants.Low to intermediate capacity steps are OK.

- Affinity chromatography

- SEC (size exclusion chromatography)

- Sedimentation through density gradients (centrifugation)

-

- Preparative PAGE

- HPLC (high pressure liquid chromatography)

Target proteins often over 95% homogeneous after polishing.

-Separation based onsize and shape.

- epen ng on s ze,proteins partitionwithin the matrix poresor outside.That determines theirelution time.

- .



Molecular weight of native globular proteins can


Consider the properties of your protein!

Is there a unique feature of your protein that will facilitate its purification?

Calmodulin is a protein that undergoes a large-scale conformational change uponcalcium binding. The two conformations have different surface charges.

Cell extract containing calmodulin

Load extract on to phenyl-sepharose column (reverse phase) and

collect flow-thru. The flow-thru contains calmodulin as well asman other roteins .

Add calcium to the flow-thru and re-load onto a phenyl-sepharose

Ca +

coumn t at as een equ rate n a u er t at conta ns cac um.

Calmodulin now binds to the column, while all other calcium-

Ca-freeBuffer

insensitive proteins flow-thru.

- .

Protein is > 95% pure!!


Several tests are required in order to establish the quantit and purit ofa protein, and its structural/functional integrity.

Quantity: UV absorbance, colorimetric assays.

Purity: Amino acid composition analysisSDS-PAGE analysis, 2D-PAGE, HPLC

Structural integrity: Western blotting, immunoprecipitationN- and C-terminal microsequencingMass spectrometry

Functional integrity: Assay for specific activity (U/mass)



Protein quantitation: UV absorbance

x: molar absorptivity (extinction

coefficient at wavelen th x.Tryptophan and tyrosine have strong

UV light absorbance in the 270-290

nm range. Because of this, The UV

absorbance of a protein containing

Tyr/Trp residues can be used to

quantitate it.

280 o yr, rp an ys- ys on s:

Native 6 M Gdn.HCl

Trp 5500 5690

C-C 125 120280=280W(nW)+280Y(nY)+280CC(nCC)

And:

[P]=OD280 / 280 (M)

Several assays for peptide bond detection using Biuret’sreaction: Bicinchoninic acid is sensitive and reliable .

Read at 562 nm

no er popuar way o measurng pro e n concen ra ons mon orsbinding of the Coomasie G250 dye to polypeptides (Bradford’sassay).

Read at 595 nm

Purit : amino acid com osition anal sis

-Protein sample digested overnight inHCl to release individual amino acids.

- m no ac s are t en coup e toninhydrin and separated by reverse-phase or ion-exchange chromatography.

-Detection of peaks at 450 nm. Relativeamounts of each amino acid obtainedfrom integration of peak areas.



SDS-PolyacrylamideGel electrophoresis

- ro en separa on ase on

polypeptide size (gel acts asmolecular sieve).

- sor e ma es pro e nsmigrate to anode when current isapplied.

- ro e ns v sua ze y s a n ngwith pigments, silver orfluorophores.

2-Dimensional PAGE: a powerful tool for mapping

N-terminal microsequencing’

1. Derivatize free amino groupof polypeptide with phenyl-isothiocyanate (PITC).

2 Acid cleava e ields the.phenylthiohydanthoin (PTH)derivative of the N-terminalamino acid.

3. The PTH-amino acid isidentified by its elution timein reverse phase HPLC.

Mass spectrometry:



Functional integrity: activity assays

Questions to ask:Questions to ask:

1. Does the final yield reflect the protein yield?

2. For recombinant proteins, is the kcat/Km similarto the one observed with native enzyme?

3. Are there any partners, prosthetic groups or co-factorsmissing?

4. Has the sensitivity to inhibitory compounds beenmaintained?

Protein Purification Lecture (Ostap)

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