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Experimental Approaches in Molecular and Cell BiologyProtein Purification and Characterization

Hanna S. YuanSept. 6, 2021

Topics: Protein Expression and PurificationProtein expressionProtein biochemical and biophysical propertiesChromatographic methods

Protein CharacterizationProtein quantificationElectrophoresisLight scattering (LS)Circular dichroism (CD)Mass spectrometry

1. Protein Expression

2. Protein Purification

3. Protein Characterization

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Main Steps in Protein Biochemistry

Biochemical methodsBiophysical methodsStructure determination

Garbage In

Garbage Out

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Protein expression is usually the bottleneck

Cloning

Expression

Purification

Crystallography

DNA

Enzymology

• Protein Biochemistry– soluble active proteins– 0.1-1 mg of protein

• Structural characterization (Crystallography)– soluble active proteins – 1-100 mg of protein

Different protein expression systems

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¡ Gene of interest is cut out with restriction enzymes (RE)

¡ Host plasmid (circular chromosome) is cut with same REs

¡ Gene is inserted into plasmid and ligated with ligase

¡ New (engineered) plasmid inserted into bacterium (transform)

Basic elements of E. coli expression systems

R: Repressor

P: Promoter

SD: Shine Delgarno sequence

(Ribosome binding site- start of mRNA)

(TT: terminator (stabilizes mRNA))

-35 -10 STOP codon

TTGACA(N)17TATAAT START codon UAU

UAAGGAGG(N)8 AUG (91%) UGA GUG (8%) UAG

UUG (1%)

R P SD coding sequence

E. coli expression vectors:

contain:

• E. coli expression elements

•Unique cloning sites

•An origin of replication

•A selectable marker

TT

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¡ Promoters§ arabinose systems (pBAD), phage T7 (pET), Trc/Tac

promoters, phage lambda PL or PR¡ Tags

§ His6 for metal affinity chromatography (Ni)§ FLAG epitope tage DYKDDDDK§ CBP-calmodulin binding peptide (26 residues)§ E-coil/K-coil tags (poly E35 or poly K35)§ c-myc epitope tag EQKLISEEDL§ Glutathione-S-transferase (GST) tags§ Celluluose binding domain (CBD) tags

Choosing a vector

The pET11 vectors (Novagen) with T7/lacO promoter

PI lacI

lacUV5 T7 polymerase

• The target protein is under control of the T7 promoter.

• Bacteriophage T7 RNA polymerase, unlike E. coli RNA polymerase, is not inhibited by

rifampicin. Thus host genes can be turned off.• T7 RNA polymerase in the bacterial

chromosome is controlled by a lacUV5 promoter.

• The T7 promoter is fused to the plac operator.• The lac I repressor inhibits expression of T7

polymerase and the heterologous protein.• IPTG is used for induction.

T7 lacO target protein T7 terminator

A copy of the lacI gene (also found in the genome) is inserted on the plasmids to achieve sufficient repressor.

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Pl lacI Plac lacO lacZ lacY lacAlacI operon lac Operon

lac repressor Beta-galactosidase Beta gal-(cleavage of lactose) Beta gal- transacetylase

permease (function?)(import of lactose)

The lac operon -the paradigm of expression regulation in E. coli

Pl lacI Plac lacO lacZ lacY lacA

In presence of glucose (in the absence of lactose), the lac repressor (lacI gene product) is bound to the lac operator and blocks RNA polymerase from binding.

When glucose is scarce, E. coli use lactose as the carbon source.β-galactosidase is induced to break down lactose.

In periods of glucose starvation (high level of cAMP) and presence of lactose:Lactose enters the cell and binds to the LacI repressor protein making it fall of the DNA. RNA polymerase can now bind to the lac promoter and initiate transcription.

-Lactose acts as an inducer (by removing the repressor) of transcription.-Induction is performed with IPTG which acts as a synthetic lactoseanalogue that binds the lacI gene product.

IPTG acts as a synthetic lactose to bind to Lac Repressor and induces Lac promoter

Pl lacI Plac lacO lacZ lacY lacA

Pl lacI Plac lacO lacZ lacY lacA

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BL21(DE3)pLysS (Novagen):• Increased stability of expressed protein from deficiency in proteases lon and

OmpT.• DE3 lysogen expresses T7 polymerase upon IPTG induction. The pLys plasmid

produces T7 lysozyme to reduce the basal level protein expression. Thus lower background expression of target genes with pLysS plasmid.

Choosing the host cell for inducible protein expression

Host strain chaperone tRNAs Inducer Resistant Marker

JM109

XL1blue

M15 Kan

B834(DE3)

BL21(DE3) pLysS IPTG Cm

BL21-CodonPlus(DE3)-RIPL (AGA, AGG, AUA, CCC,and CUA) IPTG Cm/Strep

Rosetta 2(DE3) pLysS(AGA, AGG, AUA, CUA, GGA, CCC, and CGG)

IPTG Cm

B834(DE3) -pG-KJE8dnaK-dnaJ-grpEproES-groEL

L-arabinoseTetracyclin

Cm

Host Strain Examples

BL21(DE3) strainlon and ompT proteases deficientCarries a lambda DE3 lysogen, the lacI gene and lacUV5-driven T7 RNA polymerase

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How to express multi-protein complexes

Common problems encountered with E. coli expression system

Recombinant proteins in the inclusion body:• Re-fold protein from inclusion body using urea or Guandine-HCl• Decrease the temperature in protein expression

Low protein expression level:• Using E. coli host strains with rare tRNA• codons-BL21 (DE3) RIPL• Codon optimization

Other things to try:• Tight regulation of expression.• Co-expression of molecular chaperones (protein specific)• Fusion moiteties may increase folding, solubility and resistance to proteolysis• Use of protease deficient E. coli strains.• Use of thioredoxin reductase (trxB) or glutatione reductase double mutants for expression of proteins with disulfide bonds.• Periplasmic expression.

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Baculovirus Insect cell protein expression system

¡ Promoters§ polh (polyhedrin) and p10 promoter

¡ Host strains§ Sf9 (baculovirus production/protein production)§ Hi5 (protein production)

Know your proteinPhysical Properties

a) Does it aggregate?b) Does it adhere to columns or membranes?c) How long is it stable after isolation?d) Dose it function as a monomer, dimer or trimer?

Chemical Propertiesa) known internal disulfide linkages?b) known binding partners or cofactors?c) are metal ions a part of the active protein?

What is known about the protein's stability and solubilitya) What's necessary to keep it stable and alive?b) Temperature?c) Additives (Metal ions, EDTA, DTT, K vs. Na .....)?d) Known destabilizing chemicals and conditions?

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¨ A centrifuge uses centrifugal force (g-force) to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis.

¨ The soluble fraction of the homogenate is the supernatant that remains after the other materials have been pelleted out in previous rounds of centrifugation. Within this fraction soluble proteins are found.

Protein extraction by centrifugation (Cell fractionation)

Proteins can be purified according to certain properties they possess. These properties allow us to employ different techniques in purifying proteins.

Protein Property TechniqueSolubility Differential precipitation

Size Gel filtration chromatography

Charge Ion exchange chromatography

Affinity Affinity chromatography

Polarity Reversed phase chromatography

Protein Purification

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Each protein has a different solubility so this is a method to isolate groups of protein. Ammonium sulfate is most commonly used.

Differential precipitation

Ionic strength of the medium: µ = ½ S cjzj2

Salting in: Increase of protein solubility at low ionic strengths than in pure water. Salting out: Decrease in solubility at high ionic strengths.

0.2 M 0.4 M 0.6 M

Salting out

cj is the molar concentrationszj is charge number

¡ In column chromatography an adsorbent (stationary phase) is placed in a glass tube.

¡ A protein mixture (mobile phase) is passed into the column and binds to the adsorbent.

¡ By proper choice of the eluting buffer, specific proteins can be eluted from the adsorbent and separated from other proteins in the mixture.

¡ By repeating this procedure with several different adsorbents, pure protein can be obtained.

Lehninger Principles of Biochemistry, Third Edition

Column Chromatography

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Protein can be separated by this method based on their affinity for specific groups or compounds.

▪ Lectin affinity chromatography, concavalin A column▪ Metal Interaction chromatography, Ni column.▪ Enzyme-substrate chromtograhy, GST colum▪ Immuno-affinity chromatography, IgG column

(Protein A)

Lectin affinity chromatography

Metal Interaction Chromatography

Immuno-affinity chromatography

Affinity Chromatography

Construction: hPNP46/pET28ahPNPase: N-terminal His-tagged hPNPase (residues 46–669)Host strain: BL21-RIPLExpression condition: 18℃, 20 hours, 0.5 mM IPTG

Ni-NTA resin affinity column

F W M 4 5 6 7 8 9 10

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¡ Based on attraction betweencharged groups in protein andcolumn medium.

¡ In this technique, the beads of thecolumn have a specific charge onthem. This is a result of a moleculethat is attached to these beads.

¡ Selective absorption and elutionaccomplished by change of pH orby change in salt concentration.

Ion exchange chromatography

The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge.

Cation exchange column

Separates protein molecules according to their molecular size

Size exclusion or gel filtration chromatography

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¡ Reverse phase chromatography is a separation based on the polarity of the protein.

¡ Proteins with exposed hydrophobic region (red) will be able to bind to the immobilized hydrocarbon stationary phase; the rest simply wash off

http://www.thefullwiki.org/Proteomics/Protein_Separations_-_Chromatography/Reversed_Phase

Reversed phase chromatography

Superdex 200 gel filtration column M 11 12 13 14

Purification of hPNPase

hPNPasecrystals

Lin C. L., Wang, Y.-T, Hsiao, Y.-Y., Yang, W.-Z. and Yuan*, H. S.Crystal structure of human polynucleotide phosphorylase: Insights into its domain function in RNA binding and degradation. Nucleic Acids Res. 2012, 40, 4146-4157.

50 mM Tris (pH 8.0)150 mM NaCl

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¡ Protein quantification¡ Electrophoresis¡ Light scattering (LS)¡ Circular dichroism (CD)¡ Mass spectrometry

Protein Characterization

¡ Absorption at 280 nml Absorption of aromatic acids, tryptophan, tyrosine residuesl Advantages: Quick and useful for identification of proteins before using

a more accurate methodl Disadvantages: Disturbance by other substances with absorption at 280

nmRule of thumb: A280 = 1 corresponds to 0.5 – 2 mg/mL protein

Beer-Lambert Law: A (absorption) = ε x l x c

c (mg/ml)= (A280�mol. wt.) / (ε280 � l)

ε280=(5500 � nTrp )+(1490 � nTyr )+(125 � nS-S )nTrp, nTyr , nS-S : number of Trp, Tyr and disulfide bond

Protein Quantification

ε: the theoretical molar extinction coefficient

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¡ Lowry Assayl 2-step reaction

lFirst - Cu2+ chelate with the peptide bonds resulting in reduction of Cu2+ to Cu+

lSecond –The Cu+ can be detected with Folin Ciocalteu Reagent with absorption range of 500 –750 nm

¡ Bradford Assayl Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when

bound to arginine and lysine residuesl The anionic (bound form) has absorbance maximum at 595 nm

Protein Quantification

¡ Electrophoresis: The transport of particles by an electrical field through a solid media.

¡ The migration of a protein in a gel during electrophoresis is a function of its size/shape/charge.

¡ Polyacrylamide gel electrophoresis (PAGE): gel in apparatus formed by polymerizing acrylamide with crosslinker (bis-acrylamide).

Electrophoresis

Detection of proteins by staining with Coomassie Blue or silver stain or detect radioactivity with X-ray film.

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¡ Denatured electrophoresis -Separation of proteins based on size not charge.¡ SDS binds to most proteins probably by hydrophobic interaction. ¡ One SDS for every two AAs. Thus, each protein has a similar charge-to-

mass ratio.¡ The electrophoretic mobility of a protein on an SDS polyacrylamide gel is

related to its molecular weight.¡ A good method for determining the purity of a protein and analyze a

mixture of proteins.

SDS PAGE

¡ Separations based on native size and charge.¡ Application:

(1) changes in charge due to chemical degradation or modifications.(2) detect unfolded proteins(3) oligomers or aggregation (both covalent & nonconvalents)(2) binding events (protein-protein or protein-ligand)

e.g., Electrophoresis mobility shifting assay (EMSA)(protein-nucleic acid interaction)

Native Gel Electrophoresis

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Biochemical and catalytic properties of recombinant RNase T. (a) SDS-PAGE analysis of RNase T and E92G mutants shows that both recombinant proteins were purified to homogeneity. (b) and (c) RNase T is a homodimer as shown by dynamic light scattering and gel filtration chromatography. (d) Nuclease activity assays show that RNase T digests ssDNA more efficiently than dsDNA. (e) RNase T binds ssDNA with higher affinity than dsDNA as shown by gel shift assays.

Example: Recombinant RNase T is a functional homodimer

Nature Chem. Biol. 7, 236-243 (2011).

Estimated MW~50 kDa

¡ DLS is a technique used to determine the size distribution profile of small particles in solution.

¡ Particles in suspension undergo Brownian motion.

¡ If the particles are illuminated with a laser, the intensity of the scattered light fluctuates at a rate that is dependent upon the size of the particles.

¡ Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size (hydrodynamic radius) using the Stokes-Einstein relationship. http://ujkeb.com/facilities.html

Light Scattering For the measurement of the molar mass and size of a protein in solution.

Dynamic light scattering (DLS)

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Static Light ScatteringStatic light scattering is a technique to measure absolute molecular weight using the relationship between the intensity of light scattered by a molecule and its molecular weight and size, as described by the Rayleigh theory. In the simplest terms, Rayleigh theory says that larger molecules scatter more light than smaller molecules from a given light source and that the intensity of the scattered light is proportional to the molecule’s molecular weight.There are two ways to measure absolute molecular weight by static light scattering:

Batch measurement using a cuvetteIn combination with a chromatography instrument.

MALS (Multi-Angle Light Scattering):Because the angular dependence affects the intensity of scattered light, a multi-angle detector is used to collect the scattered light. The Debye plot is extrapolated back to 0o. The MW is calculated from the intercept and Rg(Radius of gyration) is calculated from the initial slope of the line.

Disease-linked human PNPasemutants are assembled into dimers, not trimers.

Gel filtration profiles reveal that PNPase-CHis elutes in a single peak as a trimer. whereas PNPase-Nhis elutes in two peaks. Disease-linked PNPase-Q387R and PNPase-E475G, both with a C-terminal His-tag, elute in a single peak with a smaller oligomeric form compared to PNPase-CHis.

Molecular weights of PNPase-CHis, PNPase-NHis, PNPase-Q387R and PNPase-E475G were estimated by SEC-MALS and are represented as elution profiles.

SEC-MALS: Size exclusion chromatography coupled with MALS.

Golzarroshan, B, et al, Nucleic Acids Res. 46, 8630-40 (2018).

Molecular weight of hPNPase determined by SEC-MALS

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¡ CD is a variant of absorption spectroscopy which measures the difference in absorption of left and right polarised light (in the ultraviolet) by a medium.

¡ Left polarized light will be absorbed more strongly than right polarized light (or vice-versa) if the absorbing bond is asymmetric, i.e. if it is chiral.

¡ In the far UV (180 - 250 nm) the CD of a protein is usually an asymmetric alpha-carbon on either side, hence the peptide bond transitions interact to give a CD signal which is very sensitive to secondary structure

Circular dichroism (CD)

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The CD spectra were scanned from 260 to 200 nm for three times at 25 oC for different truncated constructs of TDP-43: (A) N-RRM1 and D169G mutant; (B) N-RRM12 and D169G mutant; (C) N-RRM12-G and D169G mutant. Melting temperatures were measured at the loss of the β-strand structure at 218 nm forwild-type TDP-43 and D169G mutant. The melting points (Tm) are labeled in each melting curve.

TDP-43 D169G mutants are more resistant to thermal denaturation as monitored by circular dichroism

Chiang, C.H. et al, Sci. Rep. 6, 21581 (2016).

MASS SpectrometryMass spectrometer is a method used to measure the mass-to-charge ratio of a molecule. The measurements are made on molecules in the gas phase.

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Basic components of MASS spectrometer

In MALDI, the protein samples are placed in an acidic matrix with protons of positive charges. With a short pulse of laser light, the proteins are ionized and desorbed from the matrix into the vacuum system.

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Electrospray mass spectroscopy

M: protein massn2: number of chargesX: mass of the added group

Disadvantage: Multicharged ions are frequently formed from large biological molecules, resulting in m/zratio is too small to be observed in mass detectors. The m/z limit of measurement is normally 2000-3000.

Tandem mass spectrometry (MS/MS)

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Mass spectrometry (MS) is a technique by which you can determine the mass of a protein with remarkable precision of the order of a few hydrogen atoms.It can detect any important modification (post translational modification) or variation in a protein structure.

MASS in protein characterization

Crystal structure of EndoG/CPS-6 reveals a homodimeric conformation

CPS-6 H148A/F122A1.8 Å

EXAMPLE for MASS

Lin, J.L.J. et. al. Nucleic Acids Res. 44, 10480-10490 (2016).

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The interfacial residues of CPS-6 dimer are oxidized to disrupt dimer formation

Both reduced and oxidized CPS-6 proteins were trypsin-digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

EXAMPLE for MASS

Lin, JLJ, et al, Cell Rep. 12, 279-287 (2016).

EXAMPLE for MASS

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Dimeric EndoG is shifted to monomers under high levels of ROS, and monomeric EndoG exhibits a reduced endonuclease activity

After all the effort it takes to get your protein samples, it can be tempting to work with poor proteins in the hope that any problems can be sorted out in the data analysis. It is much more effective to spend the time sorting out the problems with the proteins instead.