Growth and Purification of Human Growth Hormone

1

Presenter Info:

Nathan Jones Cary, NC Senior Physics Major at North Carolina

State University

2

What is Human Growth Hormone (hGH )?

Protein for pharmaceutical interest 191 Amino Acids Mw 22KD Alpha-helixes mostly Study stability in different environments Use neutron scattering

3

Hard sphere fitting: estimate interaction peak

)()()()( 22 qSqPVVNqI p

ρ∆=

where

depends on particle shape

average structure factor:

d ~ λ/θ 1nm-1μm

4

2)()( qFqP =

Small Angle Neutron Scattering

Q=(4π/λ)sin(θ/2)

Neutron Scattering

0

10

20

30

40

50

60

70

80

90

hydrogen deuterium nitrogen carbon oxygen

Cro

ss S

ecti

on (

in B

arns

) Scattering Cross Sections

5

Working at IBBR and BL2

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The Process

Making a plasmid Grow cells Express the protein Lysing the cells ◦ Unfolding and Refolding

Purifying protein

7

Making the Plasmid •Gh2 gene •2.1 kb •Replicates at single position

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Cell Growth

•Inoculate 50 mL LB 50 μL Ampicillin (AMP) and 100 μL Chloramphenical media with 200 μL E. Coli cells •Let cells grow to an OD of roughly 3 and inoculate in larger 1.5 L of LB •Why 3?

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Spectrophotometry

Optical Density

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LB growth curves

11

00.5

11.5

22.5

33.5

4

0 5 10 15 20 25 30 35

OD

Time (in hrs)

OD growth 6/14

OD…

0

1

2

3

4

5

6

0 1 2 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8

OD

OD

100 mL LB inoculated

100 mL LB inoculated into 1.5 L LB

OD Standardization

12

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8

OD

Time (in hrs)

Left Induction Curve 6-15

Todd's

Induced

Other

Induced

-1

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8

OD

Time (in hrs)

Right Induction Curve 6-15

Todd's

Induced

Other

Induced

100 mL LB inoculated into 2 X 1.5 L LB

Protein Expression

Isopropyl β-D-1-thiogalactopyranoside

Viral promoter that allows for expression of genes

Not metabolized by cell

13

IPTG

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25 30

OD

Time (in hrs)

Induction Curve 6/14

100 mL LB inoculated into 1.5 L LB

IPTG Induction

14

0

0.05

0.1

0.15

0.2

0.25

0.3

50μM .1 mM 1 mM Sample1 Sample2 Sample3 Sample4 Sample5 Sample6

Con

c. In

mg/

ml

Sample name

Protein Concentrations

Series1

Sample1 Normal M9 media Sample2 M9 media + 2X Glucose Sample3 M9 media + 2X Thiamine Sample4 M9 media +2X Glucose + 2X Thiamine Sample5 M9 media + 3X Glucose Sample6 M9 media + 5X Glucose

50 μM

50 μM IPTG concentration

.1 mM

.1 mM IPTG concentration

1 mM

1 mM IPTG concentration

Protein Purification

Cell lyse ◦ Sonication ◦ French Press ◦ Chemical Lysing

Unfolding/ Refolding ◦ High urea and cysteine ◦ Low urea and more cysteine

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Protein Purification-1

B-Per ◦ Bacterial Protein Extraction Reagents

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Protein Purification-2

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Different chromatography columns Ion exchange column (IEX) Hydrophobic column (HIC) Size exclusion column

Characterization

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SDS-PAGE

•Separates proteins by molecular weight •Applies negative charge to each protein by mass. •Tracking dye added.

Where the protein is?

1: LB/ Bper/s

2: LB/ B-per/p

3: M

4: Enriched /B-per/s 5: Enriched /B-per/p

7: hGH 0.25mg/ml 8: hGH 0.5 mg/ml 9: hGH 1 mg/ml 10: hGH 2 mg/ml

4 5 6 7 8 9 10 1 2 3

M9 Optimization

Be able to have labeled protein for neutron scattering

Slower growth rates than other media Contains: ◦ Na2HPO4

◦ KH2PO4

◦ NaCl ◦ NH4Cl ◦ 1M MgSO4

◦ 1M CaCl2

◦ 20% Glucose

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To enrich: •1% Thiamine hydrochloride •1.38 mg/ml FeCl2

Growth

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OD-1 Normal M9 media

OD-2 M9 media + 2X Glucose

OD-3 M9 media + 4X Glucose

OD-4 M9 media +2X Glucose+ 2X Vitamins

Inoculated 50 ml M9 with 10 ml LB

0

1

2

3

4

5

6

7

0 5 10 15 20 25 30 35

OD

Time (hrs)

M9 OD Growth

OD-1

OD-2

OD-3

OD-4

Protein Expression

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Sample1 Normal M9 media

Sample2 M9 media + 2X Glucose

Sample3 M9 media + 4X Glucose

Sample4 M9 media +2X Glucose + 2X Vitamins

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7

Con

cent

rati

on (

mg/

ml)

Time (hrs)

Protein Concentration

Sample1

Sample2

Sample3

Sample4

Inoculated 50 ml M9 with 10 ml LB

M9 media

4 5 6 7 8 9 10 1 2 3

1: hGH 1.3 mg/ml 2: M 3: 1-Bper/P 4: 2-Bper/P 5: 3-Bper/P 6: 4-Bper/P 7: 1-Bper/s 8: 2-Bper/s 9: 3-Bper/s 10: 4-Bper/s

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Sample1 Normal M9 media

Sample2 M9 media + 2X Glucose

Sample3 M9 media + 4X Glucose

Sample4 M9 media +2X Glucose + 2X Vitamins

Where the protein is And which media to choose!

1 2 4 | 1 2 4 | M | hGH

Supernatant | pellet | Marker | purified hGH

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1 M9 media +2X Glucose+ 2X vitamins+2.5% glycerol

2 M9 media+ different inoculants

4 M9 media+2X Glucose+ 2X vitamins

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1s 1p 2s 2p 4s 4p

Con

c. (

mg/

ml)

Sample

Protein Concentration

Further Research

Use of D2O for growth and optimization Continued optimization Protein decay improvements SANS of hGH and d-hGH in different

pharmaceutical relevant formulations

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Acknowledgements

•NIST •Julie Borchers

•SURF Program

•Sheila Khodadadi

•IBBR •Todd Hoopes

•John Marino

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IXC (1)

1: B10 2: B12 3: C2 4: C4 5: C6 6: M 7: E8 8: E10 9: Refolding (2 day old) 10: hGH 0.5 mg/ml

Pooled B8-B12 4 5 6 7 8 9 10 1 2 3

IXC (2)

10: Load 9: Wash 8: M 7: A8 6: A10 5: A12 4: B2 3: B4 2: B6 1: B8

4 5 6 7 8 9 10 1 2 3

HIC (1)

1: Load 2: P-column (after 1 day) 3: Wash 4: M 5: A2 6: A10 7: B1 8: B4 9: B6 10: B8

4 5 6 7 8 9 10 1 2 3

HIC (2)

1: M 2: B10 3: B12 4: C1 5: X1 6: X2 7: Q- column (after 3 days) 8: spill!! 9: Std/ 0.3 mg/ml 10: Std/ 0.6 mg/ml

Pooled B8-B12

4 5 6 7 8 9 10 1 2 3