Boston Presentation

2014 Michigan Synthetic Biology TeamUniversity of Michigan

Ann Arbor, MI

ScFV Antibody Secretion in E. coli

Eukaryote Prokaryote

Purifying Mammalian Proteins in E. coli is Problematic

Prokaryotic Periplasm mimics the Reducing Environment of ER

E. coli OsmY Secretes into the Periplasm

Utilization of OsmY as a Secretion-Based Protein Purification Method

Human Practice:Project Implications

Antibodies and the Economy

Meeting with Covance to Determine Market Viability for Antibody Detection

• Fluorescence Microscopy of Secreted mCherry

• Western Blot Analysis of Secreted mCherry

• Western Blot Analysis of Secreted ScFV Antibody

Results

Expression Vector Creates Functioning Polypeptide

Expression Vector is Capable of Secreting Protein Fusion

Functional Antibodies can be Secreted from the Construct

Future Directions• Optimize overexpression conditions for construct

• Perform ELISA with antibody-rich supernatant

• Compare OsmY construct with industry standards

• Identify effects of cleaving OsmY from ScFV on binding efficiency

• Quantify construct secretion rates, providing models with data (ELISA)

Modeling

[C]=k-1k1[P]+Sk1(1-e-k1t) [P]=1k-1+k2(k1[C]+k-2[M]) [M]=1k-2+D[M](k2[P]) [P]=[C]k1(k-2+D[M])k-1(k-2+D[M])+k2D[M] [P]=S(1-e-k1t)(k-2+D[M]k2D[M]) [M]=SD[M](1-e-k1t)

[C]=S(k-1(k-2+D[M])+k2D[M]k1k2D[M])(1-e-k1t) [C]ss=S+k-1[P]ssk1 [P]ss=k-2[M]ss+k1[C]ssk-1+k2 [M]ss=k2[P]ssk-2+D[M] [P]ss=S(k-2+D[M])k2D[M], k1(k-1+k2)(k-2+D[M])0[M]ss=SD[M] [C]ss=S(k2D[M]+k-1(k-2+D[M])k1k2D[M])

Modeling:Secretion of OsmY-Fusion

Solving the System

Assumptions:• Volume of Cytoplasm, Periplasm, and Media are constant

• Synthesis rate is independent of time

• Transfer reactions are first order

• Periplasm and Cytosol quickly reach a steady state

Secreted Protein Concentration:Predicting the Steady State

Next Step: Test and Develop

Perform ELISA,Get Data

Compare Model to Data

Adjust ModelPredict new outcomes

We Can Check One Assumption Now:

[P] and [C] quickly reach a steady state

Mol L

Time

Future Directions• Quantify periplasmic and media

concentrations of OsmY-Fusion via ELISA• Utilize ELISA data to adjust parameters,

improving robustness of models

Monetization: The Real Driving Force

• OsmY can be used to secrete diverse proteins

• We can predict how such proteins will secrete

• OsmY can be used in a bioreactor

• Useful tool for industries

STEP 1: MAKE PROTEIN

SECRETION SYSTEM

Step 2:Predict rate of Synthesis and

Secretion

STEP 3: PROFIT

Carnegie Mellon Regional Meet-Up

Collaborations

Outreach

OutreachOutreach

Acknowledgements

University of Michigan• Marcus Ammerlaan, Ph.D, Advisor• Anuj Kumar, Ph.D, Advisor• Kaitlin Flynn, Advisor• Victor DiRita, Ph.D• James Bardwell, Ph.D• Ursula Jacob, Ph.D• Ming Liu, Ph.D

TU-Braunschweig• Michael Hust, Ph.D

Covance• Christine Gwinn, Microbiology Supervisor

Thank You, Sponsors!

Acknowledgements