a functional protein chip for pathway optimization and in vitro metabolic engineering gyoo yeol jung...
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A Functional Protein Chip for Pathway Optimization
and in Vitro Metabolic Engineering
A Functional Protein Chip for Pathway Optimization
and in Vitro Metabolic EngineeringGyoo Yeol Jung and Gregory Stphanopoulos
Presentation by Hang Chau and Hoa Trinh
Gyoo Yeol Jung and Gregory Stphanopoulos
Presentation by Hang Chau and Hoa Trinh
??In the TREHALOSE PATHWAY, two enzymes are maintained at an optimal ratio - what is that ratio and what are the enzymes?
In the TREHALOSE PATHWAY, two enzymes are maintained at an optimal ratio - what is that ratio and what are the enzymes?
Presentation OverviewPresentation Overview
1. Goals of the paper2. Making protein chips
a. Preparing capture DNAb. Preparing fusion molecules
3. Model system: sequential reaction catalyzed by NDK and luciferasea. Efficiency of cross-linkingb. Measuring activity of enzymesc. Hybridization specificity
4. Trehalose pathway optimization5. Benefits of a functional protein chip
1. Goals of the paper2. Making protein chips
a. Preparing capture DNAb. Preparing fusion molecules
3. Model system: sequential reaction catalyzed by NDK and luciferasea. Efficiency of cross-linkingb. Measuring activity of enzymesc. Hybridization specificity
4. Trehalose pathway optimization5. Benefits of a functional protein chip
Why did they write this paper?
Why did they write this paper?
Pathway optimization vs. a. single gene expression
b. over-expression of all pathway genes c. Mathematical methods
Pathway reconstruction using RNA displaya. Roberts and Szostak - mRNA-protein fusions
b. Weng et al. - building “protein microarry”
Pathway optimization vs. a. single gene expression
b. over-expression of all pathway genes c. Mathematical methods
Pathway reconstruction using RNA displaya. Roberts and Szostak - mRNA-protein fusions
b. Weng et al. - building “protein microarry”
Previous shortcomings in pathway optimization Need all the enzymes and their best activity conditions a. Enzymes may not be available b. Proteins can be fragile c. Difficult to reflect optimal conditions
Previous shortcomings in pathway optimization Need all the enzymes and their best activity conditions a. Enzymes may not be available b. Proteins can be fragile c. Difficult to reflect optimal conditions
Making “protein microarray”
Making “protein microarray”
Process CStep 4 Incubation - hybridization Step 5 Wash residual fusion molecules
Process CStep 4 Incubation - hybridization Step 5 Wash residual fusion molecules
Step 1 Amplification of genes using PCR
Process A Spotting DNA on microplate
Process A Spotting DNA on microplate
Process BIn-vitro translation of mRNA-enzyme fusion molecules
Process BIn-vitro translation of mRNA-enzyme fusion molecules
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Step 1 - Amplifying genesStep 1 - Amplifying genes
Primers used for PCR Primers used for PCR
Amplify Genes of interest in-vitro transcription in-vitro translation
Step 2: Capture DNA on microplateStep 2: Capture DNA on microplate
1. Glass plate is cleaned, treated with an aminosilane, and then functionallized with a homobifunctional coupling agent phenylene 1,4-diisothiocyanate
2. Transfer capture DNA onto glass plate
3. Incubation, blocking, and washing
1. Glass plate is cleaned, treated with an aminosilane, and then functionallized with a homobifunctional coupling agent phenylene 1,4-diisothiocyanate
2. Transfer capture DNA onto glass plate
3. Incubation, blocking, and washing
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PROCESS A
Step 3: Synthesis of fusion molecules
Step 3: Synthesis of fusion molecules
Copyright ©1997 by the National Academy of Sciences
Roberts, Richard W. and Szostak, Jack W. (1997) Proc. Natl. Acad. Sci. USA 94, 12297-12302
Roberts, Richard W. and Szostak, Jack W. (1997) Proc. Natl. Acad. Sci. USA 94, 12297-12302
1. Couple protected form of Puromycin with CPG
2. Use for synthesis of an Oligonucleotide linker w/ 3’ terminal puromycin 3. Ligate the linker to 3’ end of Synthethic mRNA template TRANSLATION 4. Dissociation of ribosome
5. Purification of fusion molecule
1. Couple protected form of Puromycin with CPG
2. Use for synthesis of an Oligonucleotide linker w/ 3’ terminal puromycin 3. Ligate the linker to 3’ end of Synthethic mRNA template TRANSLATION 4. Dissociation of ribosome
5. Purification of fusion molecule
Process B
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Step 4: Fusion molecules + Capture DNA
Step 4: Fusion molecules + Capture DNA
Add mRNA-protein fusion molecules onto the DNA micro chip
Allow for hybridization at room temperature Rinse away unhybridized materials
Add mRNA-protein fusion molecules onto the DNA micro chip
Allow for hybridization at room temperature Rinse away unhybridized materials
Catalyzed reactionCatalyzed reaction
ATP + D-luciferin + O2 ADP + oxyluciferin + light
(catalyzed by luciferase)
ATP + D-luciferin + O2 ADP + oxyluciferin + light
(catalyzed by luciferase)
Model System
ADP + GTP ATP + GDP(catalyzed by NDK)
Control 1: Cross-linking efficiency
Control 1: Cross-linking efficiency
Fluorescein-labeled oligonucleotides coding luciferase capture DNA
Efficiency: ~ the ratio of the fluorescence in the well after cross-linking : fluorescence of total DNA added to well.
Conclusion: ~100% cross-linking efficiency up to 100g of capture DNA
Fluorescein-labeled oligonucleotides coding luciferase capture DNA
Efficiency: ~ the ratio of the fluorescence in the well after cross-linking : fluorescence of total DNA added to well.
Conclusion: ~100% cross-linking efficiency up to 100g of capture DNA
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Control 2: Luciferase Activity of captured
molecules
Control 2: Luciferase Activity of captured
moleculesQuickTime™ and a
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There is no significant lost in activity of the hybridized luciferase fusion molecule in comparison with luciferase enzyme in solution.
There is no significant lost in activity of the hybridized luciferase fusion molecule in comparison with luciferase enzyme in solution.
Control 3: Optimal Amount of Capture DNA
Control 3: Optimal Amount of Capture DNA
40l of fusion molecules were loaded in each wells Increasing amounts of capture DNAs Effect: Enzymatic activities increase with increasing amounts of
capture DNAs until saturation point.
40l of fusion molecules were loaded in each wells Increasing amounts of capture DNAs Effect: Enzymatic activities increase with increasing amounts of
capture DNAs until saturation point.
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• 0.3g of capture DNA molecules spotted in each well• Increasing amounts of fusion molecules• Effect: A linear relationship between enzymatic activities and increasing amount of fusion molecules up to 40l.
Control 4: Specificity of fusion
molecules to capture DNA Control 4: Specificity of fusion
molecules to capture DNA Fusion molecules of both enzymes
are present in solution
Luciferase: varying amounts of luciferase capture DNAs and constant amount of NDK capture DNAs
NDK: varying amounts of NDK capture DNAs and constant amount of luciferase capture DNAs
Linear dependence in both cases is evidence of specificity of hybridization
Fusion molecules of both enzymes are present in solution
Luciferase: varying amounts of luciferase capture DNAs and constant amount of NDK capture DNAs
NDK: varying amounts of NDK capture DNAs and constant amount of luciferase capture DNAs
Linear dependence in both cases is evidence of specificity of hybridization
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Background: TrehaloseBackground: Trehalose
It is synthesized in yeast and has also been observed in bacterial fermentation.
It is synthesized in yeast and has also been observed in bacterial fermentation.
A disaccharide made of two glucose molecule A disaccharide made of two glucose molecule Used as a multifunctional sweetener, moisture retainer in cosmetics, and preservative in pharmaceutical products and frozen foods
Used as a multifunctional sweetener, moisture retainer in cosmetics, and preservative in pharmaceutical products and frozen foods There is an ongoing research to find a way to exploit trehalose’s ability to stabilize protein for treating Huntington’s disease.
There is an ongoing research to find a way to exploit trehalose’s ability to stabilize protein for treating Huntington’s disease.
Trehalose Synthesis Pathway
Trehalose Synthesis Pathway
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Systematic optimization of trehalose synthesis pathwaySystematic optimization of trehalose synthesis pathway
A. 3g of capture DNAB. 4g of capture DNAC. 5g of capture DNA
optimal concentration of OtsA (4g)
D. 6g of capture DNAE. 7g of capture DNA
optimal concentration of PGM (6g)
F. 8g
A. 3g of capture DNAB. 4g of capture DNAC. 5g of capture DNA
optimal concentration of OtsA (4g)
D. 6g of capture DNAE. 7g of capture DNA
optimal concentration of PGM (6g)
F. 8g
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Maintaining an optimal profile of enzymatic
activities
Maintaining an optimal profile of enzymatic
activities
a. Increasing amount of capture DNA with fixed ratio of 3/2 for PGM and OtsA
b. Remaining enzymes were saturated at 8g.
ability to reconstruct pathways?
a. Increasing amount of capture DNA with fixed ratio of 3/2 for PGM and OtsA
b. Remaining enzymes were saturated at 8g.
ability to reconstruct pathways?
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1. Microarrays for protein capture for analytical applications 2. Screening of protein libraries for binding to target
molecules 3. Screening of peptides or natural products for inhibition of
binding activity 4. Reconstructing entire pathways
1. Microarrays for protein capture for analytical applications 2. Screening of protein libraries for binding to target
molecules 3. Screening of peptides or natural products for inhibition of
binding activity 4. Reconstructing entire pathways
Advantages
1. Single step of in-vitro translation 2. Processing time is minimized
Advantages
1. Single step of in-vitro translation 2. Processing time is minimized
Future Benefits Future Benefits
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