September 13, 2010

Michael Ostap Mi h l O t Office: B40 Anat-Chem Email: ostap@mail.med.upenn.edu Phone: 215-573-9758 215 573 9758

Methods for the manipulation of p p proteins: Expression Purification Detection/Quality control

The Reductionist Approach: 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.

Key questions:
Key K questions: ti

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

What organism? Which tissue? Which subcellular compartment? How much Ho m ch protein will be needed? ill

3) Is the protein soluble or membrane-bound? 4) I solubility i aqueous b ff Is l bilit in buffers i important? t t? 5) Will there be a need for:
site directed mutagenesis? domain dissection? chimeras?

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 p pp p purification scheme?

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)

When should a recombinant protein be used: 1. Native protein is present in low abundance. 2. P t i hard to 2 Protein h d t purify f if from native sources. ti 3. Removal of non-essential features desirable. 4. Genetic analysis desirable.

3. Expression of recombinant protein in heterologous f systems is problematic.

(Multisubunit complexes becoming less of a problem)

Recombinant protein production

1. Obtain a cDNA clone of your protein of interest

Recombinant protein production Step 1: Obtaining a DNA clone p g 1. Cloning by internet: clone is obtained from another lab or purchased from a bank or a company.

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

3. Optimize expression p p

4. 4 Purify the protein

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5. Protein characterization and quality control

Recombinant protein production p g Step 1: Obtaining a DNA clone 2. PCR cloning: A pair of PCR primers are designed to match the regions of the target gene encoding the N- and C-termini of the protein to be made. The obtained PCR product is then subcloned into an appropriate plasmid.

Recombinant protein production p g Step 1: Obtaining a DNA clone 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) with a hybridizing DNA/antibody probe. Screen w/probe Isolate clone & recover plasmid

PCR/RT-PCR

PCR product

Sequence& q subclone Replica filter

cDNA library/ p y polyA mRNA

Note: Full-length proteins or truncates can be generated by this method. Master plate w/colonies

Sequence-check plasmid insert

Recombinant protein production Step 2: Subcloning for overexpression p g p 1. Choice of organism:

time-money-amount-quality

Eukaryotic systems provide native post-translational processing.

Expression System
Proteolytic Cleavage G ycosy a o Glycosylation Secretion Folding Phosphorylation Acylation Amidation Percent Yield

Insect Cells

E. Coli

Yeast Cells Mammalian Cells

Prokaryotic systems: Fast, cheap, high throughput

- Escherichia coli: Most popular, best for cytoplasmic popular proteins. Variety of overexpression vectors.

Eukaryotic systems: Expensive, laborious, high fidelity

- Yeast: Simplest system with secretory pathway. Will glycosylate proteins. Disulfides will form. - I Insect cells: S t ll Secretory pathway. More elaborate glycosylation t th M l b t l l ti patterns. Disulfides will form. High levels of expression. - Mammalian cells: High fidelity for postranslational od cat o s and atu at o e y expensive. modifications a d maturation. Very e pe s e

+/+ + +/+/ + + + 1-30%

+/+/+/+/ 15%

+/+ + +/+/ + + 1%

+ + + + + + + < 1%

Recombinant protein production p g p Step 2: Subcloning for overexpression 2. Choice of vectors: Once the choice of organism is made, an appropriate expression vector must be selected:
Promoter: Constitutive or inducible? Strong or weak? Selectable marker: non-overlapping with other plasmids. non overlapping 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 compatible y restriction endonuclease ends, or by DNA recombination.

The pET system: a phage T7 RNA polymerase-driven inducible system ( y p p y (very popular for expression in E. coli) p )
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 in E. coli (disulfide-containing proteins) (disulfide containing
-Role of thioredoxin in protein solubility not entirely understood. -For some secreted proteins, mutant strains with altered cytoplasmic redox balance promote disulfide formation and correct folding (tied to solubility).

Baculovirus-mediated insect cell expression (including virus free systems) virus-free - Glycosylation patterns closer to mammalian cells than yeast. - Several other post-translational modifications available. - Moderate to high yields. - Insect cell lines Spodoptera frugiperda (Sf9) 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 constitutive protein production t i d ti

Drosophila melanogaster cells: alternate route to more popular Sf9 and T.ni cells T ni

Mammalian expression vectors: pcDNA family

- Ideal in terms of post-translational modifications - Expensive difficult to scale up Expensive, scale-up. - Low to moderate yields for most proteins.

Amplification factor given by multiple plasmid copies integrated in cell s genome cells

Vectors for secreted proteins

- Signal peptide-containing vectors allow for efficient RER insertion. - Transmembrane domains allow for protein retention at the cell surface.

Vectors including specialized signals allow for targeting to t specific i t ifi intracellular compartments ll l t t

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

Tackling complexes: dual expression from one vector

Tet responsive element Mutated form

RETROVIRAL EXPRESSION
-Highest levels of transduction. g -Strong expression.

Recombinant protein production Step 3: Protein overproduction p p

Once cells are transfected with the recombinant plasmid, optimal conditions for overproduction are sought: -What is the effect of time? -Are inducers required (IPTG Ara Tet)? At what levels? Are (IPTG, Ara, -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 production p p Step 3: Protein overproduction

Recombinant protein production p p Step 4: Protein purification

Scale-up: The size of a preparative run will depend on specific needs, the expression/purification final yield per liter of culture and the setup used for culturing. - High yields (40-400 mg protein/liter) may be obtained using affinity-tagged proteins in high expression systems (pET, ) baculovirus) - Fermentation setup results in higher yields because of improved diffusion of oxygen and nutrients into the culture, allowing for much higher cell densities densities.

The objective is to isolate the target protein from unwanted contaminants as swiftly and efficiently as possible.

How much to purify?: Functionally pure vs homogeneously pure

Critical steps in any purification scheme: 1. Capture 2. Separation 3. Polishing 3 P li hi

1. Capture

2. Separation

The objective is to separate the target protein from the bulk medium or cell lysate to prevent degradation - Inactivation of proteases (protease inhibitors) - Removal of nucleic acids (DNAse I, PEI) - Affinity tags (polyHis, FlagTag, GST, MBP) - Precipitation steps (salting out: ammonium sulfate) - Addition of stabilizers (osmolytes, redox reagents) This step must be compatible with handling large quantities of material in relatively short times.

The objective is to remove most contaminants from the preparation. Time is critical depending on the stability of the target protein. -> Medium to high capacity chromatographic steps: ion exchange (anion/cation exchangers), dyes, affinity ligands, hydroxylapatite, HIC. -> Design purification scheme so as to minimize sample preparation between steps (avoid dialysis).

ION EXCHANGE CHROMATOGRAPHY

- Proteins bind to a charged matrix by exchange with counter-ions. - Bound proteins elute from matrix by increasing the ionic strength of the mobile phase in a step-wise or continuous fashion. [Salt] [S lt] A280

AFFINITY CHROMATOGRAPHY
- A ligand (substrate, antibody, tag, co-factor) is immobilized to a matrix. - T Target protein binds selectively to matrix. Eluted with high i i strength t t i bi d l ti l t t i El t d ith hi h ionic t th or competitor molecule).

Metal chelate chromatography: A simple, powerful affinity principle for tagging

Polyhistidine tails tend to stack just as bases do This allows them to stack, do. form coordination complexes with divalent Nickel attached to a matrix through a chelating agent. NTA: Nitrilotriacetic acid (chelating agent).

3. Polishing

The objective in this step is to remove minor or persistent contaminants. Low to intermediate capacity steps are OK. Maltose binding protein (MBP): Recombinant protein purified over amylose resin - Affinity chromatography - SEC (size exclusion chromatography) - Sedimentation through density gradients (centrifugation) - Isoelectrofocusing - Preparative PAGE - HPLC (high pressure liquid chromatography) Target proteins often over 95% homogeneous after polishing.

SIZE EXCLUSION CHROMATOGRAPHY (SEC)

-Separation based on size and shape. -Depending on size, D di i proteins partition within the matrix pores or outside. That determines their elution time. -Low capacity Low capacity.

Molecular weight of native globular proteins can be determined accurately by size exclusion chromatography

Recombinant protein production Step 4: Protein purification

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 upon calcium binding. The two conformations have different surface charges.

Recombinant protein production Step 5: Protein Characterization and Quality Control

Several tests are required in order to establish the q q quantity and p y of y purity a protein, and its structural/functional integrity.

Cell extract containing calmodulin

Load extract on to phenyl-sepharose column (reverse phase) and collect flow-thru. The flow-thru contains calmodulin as well as many other proteins. proteins

Quantity: UV absorbance, colorimetric assays. Purity: Amino acid composition analysis SDS-PAGE analysis, 2D-PAGE, HPLC

Ca +
Add calcium to the flow-thru and re-load onto a phenyl-sepharose column th t h b l that has been equilibrated i a b ff th t contains calcium. ilib t d in buffer that t i l i
Ca-free Buffer

, Calmodulin now binds to the column, while all other calciuminsensitive proteins flow-thru.

Structural integrity: Western blotting, immunoprecipitation N- and C-terminal microsequencing Mass spectrometry Spectroscopic properties Functional integrity: Assay for specific activity (U/mass)

Elute column with a calcium-free buffer. calcium free buffer

Protein is > 95% pure!!

Protein quantitation: UV absorbance

x: molar absorptivity (extinction coefficient) at wavelength x.

Protein quantitation: colorimetric assays

Several assays for peptide bond detection using Biurets reaction: Bicinchoninic acid is sensitive and reliable reliable.

Tryptophan and tyrosine have strong UV light absorbance in the 270-290 nm range. Because of this, The UV absorbance of a protein containing b b f i i i Tyr/Trp residues can be used to quantitate it. 280 of Tyr, Trp and Cys Cys bonds: Tyr Cys-Cys Native 6 M Gdn.HCl Trp 5500 5690 Tyr 1490 1280 C-C 125 120 280=280W(nW)+280Y(nY)+280CC(nCC) And:

Read at 562 nm

[P]=OD280/280 (M)

Protein quantitation: colorimetric assays

Purity: amino acid composition analysis y p y

Another A th popular way of measuring protein concentrations monitors l f i t i t ti it binding of the Coomasie G250 dye to polypeptides (Bradfords assay).

-Protein sample digested overnight in HCl to release individual amino acids. -Amino acids are th coupled t A i id then l d to ninhydrin and separated by reversephase or ion-exchange chromatography. -Detection of peaks at 450 nm. Relative amounts of each amino acid obtained from integration of peak areas.

Read at 595 nm

HPLC >

SDS-Polyacrylamide Gel electrophoresis

-Protein separation b P t i ti based on d polypeptide size (gel acts as molecular sieve). -Adsorbed SDS makes proteins Ad b d k t i migrate to anode when current is applied. -Proteins visualized b staining Proteins is ali ed by with pigments, silver or fluorophores.

2-Dimensional PAGE: a powerful tool for mapping proteomes and for detection of microheterogeneity

N-terminal microsequencing (Edman s (Edmans degradation)

1. Derivatize free amino group of polypeptide with phenylisothiocyanate (PITC). 2. 2 Acid cleavage yields the phenylthiohydanthoin (PTH) derivative of the N-terminal amino acid. 3. The PTH-amino acid is identified by its elution time in reverse phase HPLC. p

Mass spectrometry:

Functional integrity: activity assays Questions to ask: 1. Does the final yield reflect the protein yield? 2. For recombinant proteins, is the kcat/Km similar to the one observed with native enzyme? 3. Are there any partners, prosthetic groups or co-factors missing? 4. Has the sensitivity to inhibitory compounds been maintained?