Protein Purification: From industrial enzymes to cancer therapy

22

Protein Purification: From industrial enzymes to cancer therapy

3

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]

4

Protein Expression and Purification Series Workshop Timeline

• Introduction

• Recombinant protein expression and purification for biomanufacturing

• Dihydrofolate reductase

• Perform affinity chromatography

• Perform size exclusion (desalting) chromatography• Quantify protein concentration

• Look at SDS-PAGE results

• Look at enzyme results

• Scaling up for the BioLogic LP

5

Protein Expression and Purification Series

Option 1CentrifugationPurificationModuleOption 3

PrepackedCartridgePurificationModule Option 2

HandpackedColumnPurificationModule

Growth andExpressionModule

SDS-PAGEElectrophoresisModule

DHFREnzymaticAssayModule

PurificationModule

6

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

7

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

8

The Value of Proteins

Bovine Growth Hormone $14

Gold* $56

Insulin $60

Human Growth Hormone $227,000

Granulocyte Colony Stimulating Factor

$1,357,000

Price Per Gram

Prices in 2011 US Dollars* As of 8/14/2011

9

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

10

Biomanufacturing Defined

The production of pharmaceutical proteins using genetically engineered cells

11

Expression Choices Cell type:

• E. coli

• Yeast

• Mammalian–CHO

12

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

13

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

14

DHFR and Cancer

•DHFR inhibition or reduction disrupts nucleic acid synthesis affecting

-Cell growth-Proliferation

•Methotrexate – chemotherapeutic agent-Competitive inhibitor of DHFR-Methotrexate resistance - correlates with

amplification of DHFR genes

15

Induction

Biotech companies genetically engineer plasmids to place genes behind inducible promoters

16

Transcriptional Regulation in the pDHFR system

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Lactose IPTG

17


18


Lactose = Induced System

19

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

20

Selection Mechanism for Mammalian cells

21

Phases of growth

22

Recovery

Separation of protein from other molecules

Purification

Separation of the protein of interest from other proteins

23

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.

24

Types of Column Chromatography •Ion Exchange (protein charge)

•Size Exclusion (separates on size)

•Hydrophobic Interaction (hydrophobicity)

•Affinity:•Protein A tail of Antibodies•His-tagged metal complexes (Ni)•Glutathione-s-transferase glutathione

25

Performing the chromatographic separation

•Gravity Chromatography•Spin Column Chromatography

•Chromatography Instrumentation•Small scale•Biomanufacturing scale

(bioreactors)

26

Protein Expression and Purification Series Workflow

Streak Cells

Overnight culture

Subculture, monitor, and induce

Harvest and lyse cells

Purify

Centrifugation or Instrumentation

Analyze

27

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

28

Affinity purification

A. Label column with initials. Snap off bottom tab of column, remove cap and place in 2 ml microcentrifuge tube.

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

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

Ni-IMAC resin slurry

200 µl

1. Pour column

2. Wash resin to remove packing buffer

3. Equilibrate resin

4. Bind GST-DHFR-His

5. Elute unbound proteins

6. Wash protein bound onto the resin

7. Elute GST-DHFR-His discard

29

Affinity purification A. Add 200 µl of

distilled H2O to column

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

Distilled H2O

200 µl

1. Pour column






7. Elute GST-DHFR-His

discard

30

A. Add 500 µl of Equilibration buffer to column

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

Equilibrationbuffer

Affinity purification1. Pour column







500 µl

discard

31


B. Gently mix for 20 min.

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

Soluble fraction

600 µl

1. Pour column







32

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

33

His tags

Imidazole

N3H+-OOC

Histidine

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

34


A. Label three 2 ml tubes:

1. Pour column







“Flow through” “Wash” “ Eluate”

35 “Flow through”

“Flow through”

B. Remove yellow tip closure.

C. Place column in 2 ml collection tube labeled “Flow Through” and remove clear top cap.

D. Centrifuge for 2 min at 1,000x g. Set aside Flow Through.

1. Pour column








Keep

36

A. Place column in 2 ml collection tube labeled “Wash”.

B. Add 600 µl Wash Buffer to column.

• Centrifuge for 2 min at 1,000xg. Set aside Wash fraction.

Wash Buffer

“Wash”

600 µl1. Pour column








“Wash” Keep

37

Affinity purification1. Pour column







A. Place column in 2 ml collection tube labeled “Eluate”.

B. Add 400 µl Elution Buffer to column.

• Centrifuge for 2 min at 1,000xg. Set aside Eluate.

Elution Buffer

“Eluate”

400 µl

“Eluate” Keep

38

Recap so far….

Started with a complex mixture of all the soluble E. coli proteins along with the induced expressed human GST-DHFR-His

Soluble fraction

Purified the GST-DHFR-His away from the E. coli proteins by using the affinity of the 6 Histidine tag on GST-DHFR-His for Ni-IMAC beads

Flowthrough Wash Eluate

~600 µl ~600 µl ~400 µl

39

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

40

• Beads in column are made of polyacrylamide and have tiny pores

• The mixture of molecules is added to the column

• Large molecules move through the column quickly traveling around the beads

• Smaller molecules move through the pores of the beads and take longer to pass through the column

http://tainano.com/Molecular%20Biology%20Glossary.files/image047.gif

Principles of Size Exclusion Chromatography

41

Principles of Size Exclusion Chromatography

• The mass of beads in the column is called the column bed

• Beads trap or sieve and filter molecules based on size

• The separation of molecules is called fractionation

• Size of pores in beads determines the exclusion limit (what goes through the beads and what goes around the beads)

• Molecules are dissolved in a buffer

42

SizeExclusion

43

Size exclusion purification(desalting)

A. Label desalting column with your initials.

B. Invert column several times to resuspend gel.

C. Snap off bottom tip and place in a 2 ml collection tube.

1. Prepare SEC column

2. Desalt GST-DHFR-HIS with SEC column

44

1. Remove green top cap and 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.

D. After draining, place column in clean 2 ml tube.

E. Centrifuge for 2 min at 1,000 x g. Discard 2ml tube containing packing buffer.

discard

discard




45

A. Label new 2 ml tube “Desalted Eluate”.

B. Carefully apply 75 ul of eluate fraction directly to the center of column. Be careful not to touch resin with pipet tip.

C. Centrifuge for 4 min at 1,000 x g.

D. Repeat addition of 75 µl of Eluate fraction to column and centrifugation.

Removing the 250 mM imidazole solution by size exclusion chromatography

“Eluate”

75 µl

2 x




46

Desalted eluate ~150 µl

GST-DHFR-His

in 10 mM Tris buffer

250 mM Imidazole has been removed




47

Protein Analysis

• Determination of success of induction, lysis, and purification of GST-DHFR-His using SDS-PAGE analysis

• Measurement of concentration using the absorbance at 280 nm

• Enzymatic activity analysis

48

250

150

100 75

50

37

25 20

15

10

1 –

Pre

cisi

on

Plu

s D

ual

Co

lor

2 –

Un

ind

uce

d c

ells

3 –

Ind

uce

d c

ells

4 –

Inso

lub

le f

ract

ion

5 –

So

lub

le f

ract

ion

6 –

Co

lum

n f

low

th

rou

gh

7 –

Co

lum

n w

ash

8 –

Elu

ted

GS

T-D

HF

R-H

is

9 –

De

salt

ed G

ST

-DH

FR

-His

stan

dar

ds

Protein analysisSDS-PAGE

1. Prepare Samples

2. Prepare TGX Gel and vertical Electrophoresis apparatus

3. Load and Run Gel

4. Stain gel

5. Analyze gel

49

Protein analysis (Quantitation using A280)

Quantitation of Protein in Desalted Fraction

+100 µl Distilled H2O

Clean UV cuvette

+ 100 µl Desalted

eluateClean UV cuvette

Turn on spectrophotometer and set absorbance to 280 nm. Add 100 µl distilled H2O to clean UV compatible cuvette.

Blank spectrophotometer with distilled H2O.

Pipet 100 µl of your desalted eluate sample (GST-DHFR-His) into clean UV compatible cuvette.

Measure absorbance of sample at 280nm and record or print the value. Return sample to 2 ml tube.

50

Beer’s Law A=cl

- the molar absorptivity ((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)

Expected results

1.3 x 10-6 – 5.3 x 10-6 M

Calculate concentration of GST-DHFR-His

51

Enzyme Assay

Absorbance at 340nm

52

Enzyme Assay

A. Set up spectrophotometer for kinetics measurements at 340 nm.

B. Blanking the instrument. Add 985 µl 1x PBS to cuvette; place in instrument, read as blank. Save cuvette with PBS

C. Running the no substrate control reaction. Add 6 µl of 10 mM NADPH to cuvette containing 985 µl 1x PBS. Add 15 µl of purified, desalted GST-DHFR-His eluate to cuvette. Cover cuvette with parafilm and invert 10 times. Immediately place cuvette in spectrophotometer and begin kinetics run.

D. As run is proceeding, record absorbance value every 15 seconds for 150 seconds. Remove and save cuvette from the spectrophotometer.

+ 985 µl 1x PBS

+ 985 µl 1x PBS

+ 6 µl NADPH

+ 15ul desalted Eluate

+ 985 µl 1x PBS

53

Enzyme Assay

E. Running the enzymatic reaction with the GST-DHFR-His, NADPH (cofactor) and DHF (substrate).Note: The enzyme reaction should be prepared while standing at the spectrophotometer. The reaction occurs extremely quickly and it is necessary to place the cuvette in the spectrophotometer and start the readings as quickly as possible once the DHF has been added.

F. Add 5 µl of 10 mM DHF to the cuvette already containing 1x PBS, your GST-DHFR-His sample and NADPH. Quickly cover the cuvette with parafilm and invert 5 times.

G. Immediately place the cuvette in the spectrophotometer and begin kinetics run. As run is proceeding, record absorbance value every 15 seconds for 150 seconds. Remove cuvette from the spectrophotometer.

+10 mM DHF

54

Instrumentation

BioLogic LPDemo

BioLogic™ LP

BioLogic DuoFlow™

55

Chromatography instrumentation

1. Pump(s)

2. Detector(s)

- UV detector

- Conductivity detector

- Pressure detector

- Fluorescence detector

3. Valves

Plus associated wiring and tubing…

56

Examining a chromatogram

BioLogic LP

57

Examining a chromatogram

BioLogic DuoFlow

58

DHFR Enzymatic Activity Calculation

Slope of Control Data x 60 = _____________ Change in Absorbance at 340 nm/minute

ΔOD, control

ΔOD, reaction

Slope of Enzyme reaction data x 60 = _____________ Change in Absorbance at 340 nm/minute

ΔOD = |ΔOD, reaction| - |ΔOD, control|

ΔC (mol/liter/min) = ΔOD ε x l

ε (extinction coefficient) = 6220 M-1 cm-1 for NADPHl (length) is the pathlength of the cuvette (usually 1 cm for most cuvettes)

59

Biomanufacturing Scaling up of the process developed during research and development

60

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

61

AVAILABLE NOW!ProteinExpressionandPurificationSeriesOrdering 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)

Protein Purification: From industrial enzymes to cancer therapy

Documents

Transcript of Protein Purification: From industrial enzymes to cancer therapy