A Functional Protein Chip for Pathway Optimization and in Vitro Metabolic Engineering Gyoo Yeol Jung...

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



Control 2: Luciferase Activity of captured

molecules

Control 2: Luciferase Activity of captured

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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.





• 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



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



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



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?



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

THE ENDTHE ENDTHE ENDTHE END

A Functional Protein Chip for Pathway Optimization and in Vitro Metabolic Engineering Gyoo Yeol Jung...

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