A Functional Protein Chip for Pathway Optimization and in Vitro Metabolic Engineering Gyoo 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?

Presentation Overview 1. Goals of the paper 2. Making protein chips a. Preparing capture DNA b. Preparing fusion molecules 3. Model system: sequential reaction catalyzed by NDK and luciferase a. Efficiency of cross-linking b. Measuring activity of enzymes c. Hybridization specificity 4. Trehalose pathway optimization 5. Benefits of a functional protein chip 1. Goals of the paper 2. Making protein chips a. Preparing capture DNA b. Preparing fusion molecules 3. Model system: sequential reaction catalyzed by NDK and luciferase a. Efficiency of cross-linking b. Measuring activity of enzymes c. Hybridization specificity 4. Trehalose pathway optimization 5. Benefits of a functional protein chip

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 display a. 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 display a. 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” Process C Step 4 Incubation - hybridization Step 5 Wash residual fusion molecules Process C Step 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 B In-vitro translation of mRNA-enzyme fusion molecules Process B In-vitro translation of mRNA-enzyme fusion molecules

Step 1 - Amplifying genes Primers used for PCR Amplify Genes of interest  in-vitro transcription  in-vitro translation

Step 2: Capture DNA on microplate 1. 1.Glass plate is cleaned, treated with an aminosilane, and then functionallized with a homobifunctional coupling agent phenylene 1,4- diisothiocyanate 2. 2.Transfer capture DNA onto glass plate 3. 3.Incubation, blocking, and washing 1. 1.Glass plate is cleaned, treated with an aminosilane, and then functionallized with a homobifunctional coupling agent phenylene 1,4- diisothiocyanate 2. 2.Transfer capture DNA onto glass plate 3. 3.Incubation, blocking, and washing PROCESS A

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, 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. 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

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 reaction ATP + D-luciferin + O 2 ADP + oxyluciferin + light (catalyzed by luciferase) ATP + D-luciferin + O 2 ADP + oxyluciferin + light (catalyzed by luciferase) Model System ADP + GTP ATP + GDP (catalyzed by NDK)

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 100  g 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 100  g of capture DNA

Control 2: Luciferase Activity of captured molecules  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  40  l 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.  40  l 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. 0.3  g 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 40  l.

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: Trehalose  It is synthesized in yeast and has also been observed in bacterial fermentation.  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

Systematic optimization of trehalose synthesis pathway A.3  g of capture DNA B.4  g of capture DNA C.5  g of capture DNA optimal concentration of OtsA (4  g) D. 6  g of capture DNA E. 7  g of capture DNA optimal concentration of PGM (6  g) F. 8  g A.3  g of capture DNA B.4  g of capture DNA C.5  g of capture DNA optimal concentration of OtsA (4  g) D. 6  g of capture DNA E. 7  g of capture DNA optimal concentration of PGM (6  g) F. 8  g

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 8  g. 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 8  g. 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. 1.Single step of in-vitro translation 2. 2.Processing time is minimized Advantages 1. 1.Single step of in-vitro translation 2. 2.Processing time is minimized Future Benefits

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