August 22, 2011

Biochemistry 201

D. Worthylake, 7152MEB, x5176

Expression & Purification of Expression & Purification of Recombinant ProteinsRecombinant Proteins

Expression & Purification of Expression & Purification of Recombinant ProteinsRecombinant Proteins

1) If you don’t have the gene that encodes the protein but you have a source, you may want to purify the protein to:

2) If you have the gene that encodes the protein, you may want to express/purify the protein for other reasons:

a) determine the amino acid sequenceb) make antibodiesc) Identification by mass spectroscopy

a) structural analysis (x-ray crystallography & NMR spectroscopy)b) enzyme functionc) Interaction partnersd) biochemistry/biophysics (phosphorylation, regulation, etc.)e) Functional studies (cellular localization by confocal microscopy, etc)f) Pharmaceutical intervention

Why express/purify protein(s)?Why express/purify protein(s)?Why express/purify protein(s)?Why express/purify protein(s)?

TOP 10 Things to considerTOP 10 Things to considerTOP 10 Things to considerTOP 10 Things to consider1) Which protein construct to express

2) Expression host (bacterial, insect cell, yeast, or mammalian)

3) Cell line for expression

4) Promoter for induction of protein production

5) Codon optimization (for mammalian proteins expressed in bacteria)

6) Cloning method

7) May require expression as a fusion protein

8) May require co-expression with molecular chaperones

9) Affinity tag(s) for purification & protease cleavage site to remove the tag

10) Purification protocol & buffer to keep protein happy and active

a) An entire protein ?If yes, don’t need to worry about limits

b) A domain from a mosaic protein ?Need to worry about limits

Protein construct and expression hostProtein construct and expression hostProtein construct and expression hostProtein construct and expression host1) Engineering of protein construct

2) Which organism to express the protein in

b) If protein is of non-bacterial origin, because of post-translational modification in non-bacterial cells, may need to express in higher organisms:

Bacteria Yeast insect cells (SF9 or Hi5) Mammalian cell lines

a) If protein is of bacterial origin, express in bacteria

(least expensive & time consuming)

(most expensive & time consuming)

c) May need to express as a fusion protein or require codon-optimization

Bacterial expression systemAdvantages – Easy, great over-expression, low protease activity, no post-translational modifications

Disadvantages – Protein solubility, lack of post-translational modifications

Eukaryotic expression systemAdvantages – Protein solubility, post-translational modifications

Disadvantages – Expensive, low yield, proteases, time consuming

Isolate protein from native sourceAdvantages – Protein solubility, authenticity

Disadvantages – Expense/effort, yield, slaughter-housesWaring blenders

Hierarchy: Bacteria, Yeast, SF9, Hela, native tissue

Choice of expression hostChoice of expression hostChoice of expression hostChoice of expression host

Bacterial host BL21(DE3)Bacterial host BL21(DE3)Bacterial host BL21(DE3)Bacterial host BL21(DE3)

b) DE3_pLysS1) DE3 strains have leaky expression, which

leads to problems if expressed protein is toxic.

2) Plasmid-encoded T7 lysozyme inhibits T7 RNAPol and decrease leaky expression.

c) Host for cloning1) These hosts lack T7 RNAPol , and so are

suitable for plasmid amplification and not protein expression.

a) DE31) Host-encoded lac Repressor represses host

RNAPol transcription of T7 RNAPol from lac promoter.

2) IPTG induction knocks lac Repressor off and allow host RNAPol to transcribe T7RNAPol.

3) T7 RNAPol transcribe gene from T7 promoter on plasmid.

4) Lon/OmpT protease deficient.

Hosts for protein expression

Figures from www.novagen.com

Isopropyl β-D-1-thiogalactopyranoside (IPTG) = allolactose mimic

Company Cell Genotype PhenotypeInvitrogen BL21Star RNaseE(131) more stable mRNA

BL21-AI ara- arabinose induction

all companies pLysS T7 lysozyme decreases leaky expression

Clontech BL21Pro TetR tetracycline induction

Lucigen C41/C43 ?? Expression of toxic proteins

Specialized bacterial cell lines for protein Specialized bacterial cell lines for protein expressionexpression

Specialized bacterial cell lines for protein Specialized bacterial cell lines for protein expressionexpression

Novagen Rosetta2 7 rare tRNA overcome codon usage deficiencyOrigami 2/B Thrx-/GluthRed- enhance formation of disulfide bonds

You cannot just mix and match, as plasmids with the same origin of replication cannot be transformed into the same cells. Most expression plasmids have pBR322 ori, which is compatible with p15A.

p15Ap15Ap15AF/p15A+Fp15A

p15Ap15A

Consideration of codon usageConsideration of codon usageConsideration of codon usageConsideration of codon usage

Basic elements of a plasmid/vector Basic elements of a plasmid/vector Basic elements of a plasmid/vector Basic elements of a plasmid/vector

pET developed by WF Studier & BA Moffatt in 1986

1) Ap = ampicillin resistance

2) ori = ColE1/pBR322 origin of replication

3) lacI = lac repressor; bind lacO until IPTG induction

4) T7P = T7 Polymerase promoter

5) lacO = lac operator where lac repressor binds

6) = multiple cloning site

1. Restriction digestion-based methods are inefficient and require that your gene of interest does not have the same internal restriction site(s) as present in MCS.

2. Gateway-based methods are powerful.

Selection of cloning method is criticalSelection of cloning method is criticalSelection of cloning method is criticalSelection of cloning method is critical

3. Ligation-independent cloning is much more effective than ligation reaction.

The green/red parts of the primers are not complementary with the gene!

Gateway technology overview Gateway technology overview (discussed for (discussed for completeness)completeness)

Gateway technology overview Gateway technology overview (discussed for (discussed for completeness)completeness)

See file invitrogen_Gatewaymanual2003.pdf (optional reading)

Gateway recombination reactionsGateway recombination reactionsGateway recombination reactionsGateway recombination reactions

Entry clone

Expressionclone

See file invitrogen_Gatewaymanual2003.pdf (optional reading)

Generating an Generating an entryentry clone cloneGenerating an Generating an entryentry clone clone

TAGG ATCC

*

TAGG ATCC

*

(Int + IHF)

(Original site) (Original site)

See file invitrogen_Gatewaymanual2003.pdf (optional reading)

Don’t put in stop codon (*) if to have a C-ter tag

Recombining Recombining entry entry and and destinationdestination clones clones to get an to get an expressionexpression clone clone

Recombining Recombining entry entry and and destinationdestination clones clones to get an to get an expressionexpression clone clone

TAGG ATCC

*

TAGG ATCC

*

(Int + IHF + Xis)

(Original site restored) (Original site restored)

See file invitrogen_Gatewaymanual2003.pdf (optional reading)

PCRPCRPCRPCR

new cumulativeCycle 1: 2o = 1 1 = 21 - 1

Cycle 3: 22 = 4 4 + 3 = 7 = 23 - 1

Cycle n: 2n-1 2n - 1

…

15 cycles = 32,767 copies20 cycles = 1,048,575 copies30 cycles = 1,073,741,823 copies

www.neb.com

and Processive and Processive DNAPolDNAPol

and Processive and Processive DNAPolDNAPol

1) decreases lower-molecular weight fragments2) decreases extension time from 1’/kb to 10-20”/kb, so a

typical amplification takes ~1hr instead of 3-4 hours.

3) NEB Phusion (dbd) & Takara Speedstar (antibody-based)

Cycle 2: 21 = 2 2 + 1 = 3 = 22 - 1

3. Ligation-independent cloning is much more effective than ligation reaction.

LIC cloning, reminderLIC cloning, reminderLIC cloning, reminderLIC cloning, reminder

2. Verify on agarose gel that gene was amplified (10) (40’)

DAY 1 (~6.5 hr)1. PCR gene from SOURCE vector (50) (80’)

LIC-Subcloning of a gene, from beginning to LIC-Subcloning of a gene, from beginning to endend

LIC-Subcloning of a gene, from beginning to LIC-Subcloning of a gene, from beginning to endend

5. Add 100ng PCR fragment to 15ng DESTINATION vector and let anneal on ice (30’)

3. Digest remaining sample with DpnI and do PCR clean-up (75’)

4. Digest DESTINATION vector and PCR fragment with T4 DNAPol to generate single-stranded overhangs (60’)

6. Transform into XL10Gold supercompetent cells (100’) and plate overnight

DAY 2 (~2.5 hr)7. Do colony PCR on 2-4 colonies to verify gene was inserted

into vector:a) pick colony with pipet tipb) resuspend in 50 steril water and vortex to mixc) take 1 for PCR to verify insert (120’)d) inoculate remaining sample in TB for overnight growthe) if gene is inserted, do miniprep (30’)f) Verify construct by DNA sequencing

2. Oswald Avery (1944)demonstrated uptake of DNA into bacteria and coined the term bacterial transformation.

1. Frederick Griffith (1928)first demonstrated by showing that non-virulent Streptococcus pneumonia could be made virulent by exposing it to a virulent strain which has been heat-shocked.

Bacterial transformationBacterial transformationBacterial transformationBacterial transformation

3. To make cell competent:Grow cells to mid-log phase and treat/wash with CaCl2 solution. The bacterial cell wall is permeabilized by Chloride ion and swells up with the uptake of water.

4. Bacterial transformation:a) Addition of plasmid DNA to cells, followed by cold- and heat-shock allows plasmid to enter

through the small holes in the cell wall. (Can also use electroporation to create pores in cell wall).

b) Amplify the number of cells in SOC media.c) Plate on LB agar (+ antibiotic) to select for transformed cells only (antibiotic resistance is

conferred by gene encoded by on plasmid).

Expression in insect and mammalian cells are expensive and time consuming. Therefore, a feasible method is required for expression in bacterial cells as a first choice.

The apparent solubilizing effect of the fusion partner may be misleading, as the purified protein can precipitate when cleaved from its fusion partner.

Selection of expression vector & fusion Selection of expression vector & fusion partnerpartner

Selection of expression vector & fusion Selection of expression vector & fusion partnerpartner

Laila Niiranen, … Nils P. Willassen, Protein Expression & Purification 52 (2007) 210-218.

Expression difficulty in bacterial cells may be overcome by expression as a fusion protein. Expression at lower temperature improves solubility.

LIC vector for LIC vector for in-vivo in-vivo cleavage from fusion cleavage from fusion proteinprotein

LIC vector for LIC vector for in-vivo in-vivo cleavage from fusion cleavage from fusion proteinprotein

In-vivo cleavage helps remove false-positive expression (protein once purified and cleaved from its fusion partner precipitates!).

Co-express TVMV protease with fusion protein, with TVMV under control of a different promoter than the one used for the fusion protein.

In-vivo In-vivo cleavage improves protein solubilitycleavage improves protein solubilityIn-vivo In-vivo cleavage improves protein solubilitycleavage improves protein solubility

Soluble

insoluble

In-vivo In-vivo cleavage helps protein purificationcleavage helps protein purificationIn-vivo In-vivo cleavage helps protein purificationcleavage helps protein purification

Intact fusion protein in-vivo cleaved proteinM L FT W E +tev L FT W E +tev L FT W L FT W

Delayed Delayed In-vivo In-vivo cleavage improves protein cleavage improves protein solubilitysolubility

Delayed Delayed In-vivo In-vivo cleavage improves protein cleavage improves protein solubilitysolubility

0hr delay

2hr delay

Trigger factor (Tf) • binds 50S subunit;• peptidyl-prolyl cis-trans isomerase

DnaJ/K• binds nascent polypeptides;• shield exposed hydrophobic patches from folding unfavorably

GrpE• binds polypeptides released from DnaK/J• releases polypeptides into folded form or shuttles to GroEL/ES

GroEL/ES• helps fold/refold proteins already in compact state but are not yet folded

Co-expression with molecular chaperonesCo-expression with molecular chaperonesCo-expression with molecular chaperonesCo-expression with molecular chaperones

Learn all you can before beginning

MSA can often give you ideas for deciding on construct limits

Even better if there’s some structural information!

If multiple sequence alignments do not help and thereisn’t any structural info, try secondary structure prediction

http://www.compbio.dundee.ac.uk/www-jpred//

..but try several startsand stops (primers arecheap!)

Do you have the gene?

Lots of output!Here’s what you want

buy

Shopping cart – price varies with order size

Before starting, confirm that you can make a significant quantity of soluble protein. Small scale solubility experiments are very

important and typically will involve varying inducer concentration, expression temperature, expression construct, etc.

Each protein is unique – must exploit differences

Particular affinities GST, 6xHis, antibodies

Solubility (NH4)2SO4, PEG precip.

Charge ion exchange

Hydrophobicity hydrophobic chromatography

Size gel exclusion

Iso-electric point iso-electric focusing

Thermal stability alter temp.

Express protein in frame with an affinity tag – often tag is removable with a protease.Common tags: 6xHis, GST, CaM, MBP. Use affinity chromatography for first step!

Nitrilotriacetic acid

Imidazole

pH 7.4

electron coordination bonds

Kirkegaard & Perry Laboratories, Inc

Nickel-affinity chromatography (Histrap)Nickel-affinity chromatography (Histrap)Nickel-affinity chromatography (Histrap)Nickel-affinity chromatography (Histrap)

If the affinity tag is removable, go back over column and collectflow-through (or digest on the column).

Ion exchange chromatography (what is the theoretical pI of your protein?)DiEthylAminoEthane (DEAE), CarboxyMethyl (CM), Quaternary amine, Sulfonic acid.

http://www.proteinchemist.com/tutorial/iec.html

These functional groups are charged over a broad pH range. Why would that be desirable?

+

++

++

+

+

+

----Na+

Cl-

Cl-

Cl-

Cl-

Na+

Na+

Na+

Na+

Elute (High salt)

Anion #2( Cl- )

YF

P

+

++

++

+

+

+

----

Bind (Low salt)

Anion #1( protein )

YF

P

pH=6

Anion exchange chromatographyAnion exchange chromatographyAnion exchange chromatographyAnion exchange chromatography

50mM NaCl

500mM NaCl

Run a 20 x (column volume) linear gradient and collect fractions

example chromatogram

Run SDS-PAGE of fractions to decide which to pool(sacrifice yield for purity?)

Trp, Tyr, Phe,disulfides

Linear gradient(also step)

Some proteins, usually larger proteins, can bind toboth anion and cation exchange matrices – change

pH to enhance interaction.

Stronger and higher resolution ion exchange media (Q, SP) may be employed to separate proteins that were not baseline

separated with weak ion exchange step.

Q column SP column

Electrostatic potential mappedonto a molecular surface

Separates proteins by size.

Your protein should elute at the proper volume for its expected MW. Want a nice, symmetric peak in the chromatogram.

Small proteins “see” abigger volume than do large proteins

Size exclusion chromatographySize exclusion chromatographySize exclusion chromatographySize exclusion chromatography

Salting out – Proteins precipitate differentially in the presenceof (NH4)2SO4 or polyethylene glycol - It’s probably worth trying

Hydrophobic – Load proteins onto phenyl sepharose in presence of ~1.5M (NH4)2SO4 and run decreasing [(NH4)2SO4]gradient. More hydrophobic elutes later.

Isoelectric focusing – Electrophorese protein in matrix containing pH gradient. When the protein reaches that pH where it has no net charge it ceases to migrate. Retrieve protein from matrix.

Some other chromatographic techniquesSome other chromatographic techniquesSome other chromatographic techniquesSome other chromatographic techniques

Day 1Transform Rosetta 2 cells with plasmid containing tvmv gene and plate overnight

Expression of TVMV proteaseExpression of TVMV proteaseExpression of TVMV proteaseExpression of TVMV protease

Day 4 (or 2)a) thaw cells from -80C and lyse by sonication or with Emulsiflex (cells are squeeze through a small pin-hole by high pressure).

b) Start purification

Day 2 (or -1)In the evening, pick a colony and grown a 10ml overnight starter culture @ 37C/200RPM.(you can save a day by inoculating from a glycerol stock.)

Day 3 (or 1)a) In morning, inoculate 2L media with overnight starter and grow @37C/200PRM.

b) After 2-4 hours (OD600 ~0.6-1), add IPTG to 0.5mM final concentration and induce for 4hrs @30C/200RPM.

Expression optimization:at mid-log phase, lower temperature to 12-20C, add lower amount of IPTG and induce overnight, or for slow leaky expression, no IPTG for 2 days (membrane proteins)

c) Harvest cells (4000 RPM/20’), resuspend in 50mL lysis buffer (+ protease inhibitors) and store in -80C.

1) Load sample in 10mM Imi (pH8)2) Wash 5CV of 10mM Imi in 1M NaCl (helps remove DNA

bound to DNA-binding proteins)3) Start gradient: a) 1030mM / 5CV (initial wash) b) 3060mM / 30CV (more stringent wash) c) 60500mM / 1CV (start elution) d) 500mM / 5CV (complete elution)

Purification of TVMV proteasePurification of TVMV proteasePurification of TVMV proteasePurification of TVMV protease

2) Superdex 75 26/601) Nickel column

Pool for S75 save

Vo (~110ml)

Injection

Purification of TVMV proteasePurification of TVMV proteasePurification of TVMV proteasePurification of TVMV protease

Anti-His6 western

Confirming expressed protein by Western Confirming expressed protein by Western Confirming expressed protein by Western Confirming expressed protein by Western

1) 30-45’/210V: run SDS-PAGE gel[SDS binds protein tightly (1 SDS/2 aa) to give equivalent q/m ratio for all proteins; hence proteins are separated based on MW]

1) 2x 15’: rinse gel in TB2) 45’/35V: electroblot (shown to the left)3) 30’: wash NC in 25ml PBST + 5% w/v fat-free milk4) 3x 5’: wash NC in 25ml PBST5) 30’: soak NC in 20ml PBST + antibody_HRP (1:20000)6) 3x 5’: wash NC in 25ml PBST each7) 5’: expose NC to substrate, 0.75 + 0.75 ml

(Pierce SuperSignal West Dura)9) Develop/image NC

~3.5 hours total

TB: 25mM Tris[8.3], 192mM Glycine)PBS: 1.54mM KH2PO4, 2.71mM Na2HPO4, 167mM NaClPBST: PBS + 0.05% v/v Tween20)

Western blottingWestern blottingWestern blottingWestern blotting

10 things you should know10 things you should know10 things you should know10 things you should know1) How BL21(DE3) cells work (host-encoded T7RNAPol and lacI repressor)

10) Basic idea of protein purificationa) nickel-affinity (Histrap)b) Size-exclusionc) ion-exchanged) western blote) hydrophobic

2) General idea about different bacterial cell lines for optimizing expression:a) different promotersb) increasing mRNA stabilityc) Lon/OmpT protease deficiencyd) Rosetta2 cells and codon-optimization

3) Basic idea of plasmid and antibiotic selection.

4) Different cloning method (ligation, LIC, Gateway)

5) How PCR works – use of processive DNAPol to save time

6) Bacterial transformation

7) Improving protein solubility by expression as fusion protein, and which partner works best.

8) in-vivo cleavage of protein from fusion partner

9) co-expression with molecular chaperones