Growth and Purification of Human Growth Hormone...What is Human Growth Hormone (hGH )? Protein for...
Transcript of Growth and Purification of Human Growth Hormone...What is Human Growth Hormone (hGH )? Protein for...
Growth and Purification of Human Growth Hormone
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Presenter Info:
Nathan Jones Cary, NC Senior Physics Major at North Carolina
State University
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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
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Hard sphere fitting: estimate interaction peak
)()()()( 22 qSqPVVNqI p
ρ∆=
where
depends on particle shape
average structure factor:
d ~ λ/θ 1nm-1μm
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2)()( qFqP =
Small Angle Neutron Scattering
Q=(4π/λ)sin(θ/2)
Neutron Scattering
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10
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hydrogen deuterium nitrogen carbon oxygen
Cro
ss S
ecti
on (
in B
arns
) Scattering Cross Sections
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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
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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
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00.5
11.5
22.5
33.5
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0 5 10 15 20 25 30 35
OD
Time (in hrs)
OD growth 6/14
OD…
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1
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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
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0
1
2
3
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6
0 1 2 3 4 5 6 7 8
OD
Time (in hrs)
Left Induction Curve 6-15
Todd's
Induced
Other
Induced
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0
1
2
3
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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
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IPTG
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0.5
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1.5
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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
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0
0.05
0.1
0.15
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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
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1
2
3
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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
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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
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0.2
0.4
0.6
0.8
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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