Pepsi bio link

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Protein Purification: From industrial enzymes to cancer therapy

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Protein Expression and Purification Series

Instructors

Jim DeKloeSolano Community [email protected]

Bio-Rad Curriculum and Training Specialists:Sherri Andrews, Ph.D. (Eastern US)

[email protected]

Leigh Brown, M.A. (Central US)[email protected]

Damon Tighe (Western US)[email protected]

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Why Teach about Protein Expression and Purification?

•Powerful teaching tool

•Real-world connections

•Link to careers and industry

•Tangible results

•Laboratory extensions

•Interdisciplinary – connects biochemistry, biomanufacturing, chemistry, biology and medical science

• Mimics a complete workflow utilized in research and industry

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Protein Expression and Purification Series

Option 1CentrifugationPurificationModuleOption 3

PrepackedCartridgePurificationModule Option 2

HandpackedColumnPurificationModule

Growth andExpressionModule

SDS-PAGEElectrophoresisModule

DHFREnzymaticAssayModule

PurificationModule

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Protein Expression and Purification Series Advantages

• Follows a complete workflow including bacterial cell culture, induction, fractionation, purification, and analysis of purified protein

•Teaches affinity purification

• Work with a non-colored protein that is comparable to real world applications

• Includes ability to run at small scale using a 16k microcentrifuge or scaling up and using chromatography instrumentation

•Possibility of extensions including western blots, ELISAs, site-directed mutagenesis studies, induction experiments

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Protein Expression and Purification Series Workshop Timeline

• Introduction

• Recombinant protein expression and purification

for biomanufacturing

• Dihydrofolate reductase

• Affinity purification

• Perform affinity chromatography

• Perform size exclusion (desalting) chromatography

• Quantitate purified protein

• Demonstration of BioLogic LP chromatography

instrument

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The Value of Proteins

Bovine Growth Hormone $14

Gold $48

Insulin $60

Growth Hormone $227,000

Granulocyte Colony Stimulating Factor

$1,357,000

Price Per Gram

*Prices in 2011 US Dollars

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Biomanufacturing Defined

The production of pharmaceutical proteins using genetically engineered cells

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Expression Choices Cell type:

•E. coli•Yeast•Mammalian

–CHO

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Expression ChoicesParameter Bacteria Yeast Mammalian

Contamination risk Low Low High

Cost of growth medium

Low Low High

Product titer (concentration)

High High Low

Folding Sometimes Probably Yes

Glycosylation No Yes, but different pattern Full

Relative ease to grow Easy Easy Difficult

Relative ease of recovery

Difficult Easy Easy

Deposition of product Intracellular Intracellular or extracellular Extracellular

Product Intracellular Often secreted into media Secreted

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PROTEIN: USED IN THE TREATMENT OF:

Cell Production

Insulin Diabetes E. coli

Human growth hormone Growth disorders E. coli

Granulocyte colony stimulating factor Cancers E. ColiErythropoietin Anemia CHO cellsTissue plasminogen activator Heart attack CHO cellsHepatitis B virus vaccine Vaccination YeastHuman papillomavirus vaccine Vaccination Yeast

Protein – The product of Biotech

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DHFR —Dihydrofolatereductase

•Converts dihydrofolate into tetrahydrofolate (THF) by the addition of a hydride from NADPH

•THF is a methyl (CH3) group shuttle required for synthesis of essential molecules

- nucleotides- amino acids

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DHFR and Cancer

•DHFR inhibition or reduction disrupts nucleic acid synthesis affecting

-Cell growth-Proliferation

•Methotrexate – one of the first chemotherapeutic agents

-Inhibits DHFR-Methotrexate resistance - correlates with

amplification of DHFR genes

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GST-DHFR-His Construct

GST – DHFR - His

Glutathione-s-transferase

•Added to increase solubility

•Can be used as a secondary purification methodology

Human dihydrofolate reductase

•Gene product of interest

•Target for chemotherapy reagents

Histidine tag

•6 Histidine tag that binds to certain metals such as nickel

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Induction

Biotech companies genetically engineer plasmids to place genes behind inducible promoters

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Transcriptional Regulation in the pDHFR system

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Lactose IPTG

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2 phases of growth

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Recovery

Separation of protein from other molecules

Purification

Separation of the protein of interest from other proteins

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Chromatography Basics

• Mobile phase (solvent and the molecules to be separated)

• Stationary phase (through which the mobile phase travels)– paper (in paper chromatography)– glass, resin, or ceramic beads (in column

chromatography)

• Molecules travel through the stationary phase at different rates because of their chemistry.

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Types of Column Chromatography •Ion Exchange (protein charge)

•Size Exclusion (separates on size)

•Hydrophobic Interaction (hydrophobicity)

•Affinity:•Protein A •His-tagged•Glutathione-s-transferase

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Performing the chromatographic separation

•Gravity Chromatography•Spin Column Chromatography

•Chromatography Instrumentation•Small scale•Biomanufacturing scale

(bioreactors)

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Protein Expression and Purification Series Workflow

Streak Cells

Overnight culture

Subculture, monitor, and induce

Harvest and lyse cells

Purify

Centrifugation or Instrumentation

Analyze

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CentrifugeRCF to RPM conversion • Accurate RCF(g) is important for

chromatography resins

• RPM to RCF varies for different models of centrifuges due to variation in rotor radius

• Determine RPM for 1,000 x g. The Bio-Rad 16K microcentrifuge rotor has a radius of 7.3 cm

RCF = relative centrifugal force

RPM = rotations per minute

R = radius in cm from center of rotor to middle of spin column

1,0003,497

7.3

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Affinity purification

Pouring a 100 µl Ni-IMAC column

Label column with initials. Prepare column. Snap off bottom tab of empty column, remove cap and place in 2 ml collection tube.

Add 200 µl of Ni-IMAC resin slurry to empty column

Centrifuge for 2 minutes at 1,000 x g. After spin, discard buffer that has collected in the collection tube.

Ni-IMAC resin slurry

200 µl

•Pour column

•Wash resin to remove packing buffer

•Equilibrate resin

•Bind GST-DHFR-His

•Elute unbound proteins

•Wash protein bound onto the resin

•Elute GST-DHFR-His

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Add 200 µl of distilled H2O to column

Centrifuge for 2 minutes at 1,000 x g. After spin, discard water from collection tube.

Add 500 µl of Equilibration buffer to column

Centrifuge for 2 minutes at 1,000 x g. After spin, discard Equilibration buffer and collection tube. The column is now ready to use.

Washing and equilibrating the 100 µl Ni-IMAC column

Distilled H2O

200 µl

Equilibrationbuffer

500 µl

•Pour column







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Gently mix for 20 min.

Place yellow tip closure on bottom of column. Add 600 µl Soluble Fraction to Column; Put on clear top cap.

Soluble fraction

Binding the GST-DHFR-His to the Ni-IMAC resin

600 µl

•Pour column







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His tags

N3H+-OOC

Histidine

Resin

• His tags are typically a series of 6 histidines added to the C or N terminus of a recombinant protein

Ni

Ni

Ni

Ni

N

NH

NN

H His-tagged Recombinant

Protein

• His tag and column interaction

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His tags

Imidazole

N3H+-OOC

Histidine

• His and imidazole structure similarities• Imidazole competes with His for Ni2+ sites

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Performing affinity chromatography

Place column in 2 ml collection tube labeled “Wash”. Add 600 µl Wash Buffer to column.

Centrifuge for 2 min at 1,000 x g.

Set aside Wash fraction.

Label three 2 ml tubes: “Flow through”, “Wash” and “Eluate”.

Remove yellow tip closure. Place column in 2 ml collection tube labeled “Flow Through” and remove clear top cap. Centrifuge for 2 min at 1,000 x g.

Set aside Flow Through.

Flowthroughfraction

Wash Buffer

Wash fraction

600 µl

•Pour column







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Performing affinity chromatography (continued)

Elution Buffer

Eluate


Set aside Eluate.

Place column in 2 ml collection tube labeled “Eluate”. Add 400 µl Elution Buffer to column.

400 µl

Flow through Wash Eluate

~600 µl ~600 µl ~400 µl

Collected fractions

•Pour column







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Size exclusion purification(buffer exchange)

Eluate fraction

GST-DHFR-His in 20 mM sodium phosphate, 300 mM NaCl and 250 mM imidazole

Imidazole

250 mM imidazole

solution has an A280= 0.2-0.4

W and Y contribute to A280 of proteins

NEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN CONCENTRATION USING A280

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SizeExclusion

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Label desalting column with your initials. Prepare desalting column by inverting sharply several times to resuspend gel

Centrifuge for 2 min at 1,000 x g. Discard remaining packing buffer and collection tube.

Snap off tip and place in 2 ml collection tube. Remove green top cap.

Allow excess packing buffer to drain by gravity to top of resin bed. If the column does not begin to flow, push the cap back on the column and then remove to start the flow. After draining, place column in clean 2 ml tube.

Preparing the size exclusion column for usage

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EluateDesalted

eluate

Label new 2 ml tube Desalted eluate. Carefully apply 75 ul of eluate fraction directly to the center of column. Be careful not to touch resin with pipet tip.


75 µl

Removing the 250 mM imidazole solution by size exclusion chromatography

Collected fraction

Desalted Eluate

~75 µl

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Protein analysis (Quantitation using A280)

Desalted eluate

Clean UV cuvette

Set absorbance to 280 nm

Blank spec with distilled H2O

Measure absorbance of sample at 280nm

Print out your data

75 µl

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Beer’s Law

A=cl

- the molar absorbtivity ((mol/L)-1 cm-1)

l - the path length of the sample (usually 1cm-cuvette)

C - the concentration of the compound in solution (mol/L)

For GST-DHFR-His

= 75,540 (mol/L)-1 cm-1

C (mol/L) = Absorbance

75,540 (mol/L)-1 cm-1 x 1 cm

Protein analysis (Quantitation using A280)

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Enzyme Assay

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Instrumentation

BioLogic LPDemo

BioLogic™ LP

BioLogic DuoFlow™

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Biomanufacturing Scaling up of the process developed during research and development

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Bio-Rad:Curriculum Training Specialists [email protected]

http://explorer.bio-rad.com

Technical Support: 1(800)4BIORAD [email protected]

Northeast Biomanufacturing Center and Collaborative (NBC2)

http://www.biomanufacturing.org

Bio-Link (Elaine Johnson, Director)http://www.bio-link.org

Jim DeKloe:[email protected]

Resources and References

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AVAILABLE SUMMER 2011ProteinExpressionandPurificationSeriesOrdering info

Option 1CentrifugationPurificationModuleOption 3

PrepackedCartridgePurificationModule Option 2

HandpackedColumnPurificationModule

Growth andExpressionModule

SDS-PAGEElectrophoresisModule

DHFREnzymaticAssayModule

PurificationModule

•166-5040EDU, Centrifugation Process Series

•166-5045EDU, Handpacked Column Process Series (instrumentation)

•166-5050EDU, Prepacked Cartridge Process Series (instrumentation)

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