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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