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A platform for engineering peptide ligase for building next generation peptide therapeutics.

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R41GM136037-01
Agency Tracking Number: R41GM136037
Amount: $225,000.00
Phase: Phase I
Program: STTR
Solicitation Topic Code: 400
Solicitation Number: PA18-575
Solicitation Year: 2018
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-02-07
Award End Date (Contract End Date): 2021-08-06
Small Business Information
San Francisco, CA 94117-3371
United States
DUNS: 079569722
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (415) 476-9819
Business Contact
Phone: (510) 813-6420
Research Institution
SAN FRANCISCO, CA 94143-0962
United States

 Nonprofit College or University

There is an increased interest in peptide medicines in pharmaceutical research and development
(Randamp;D) because peptides are recognized as highly selective and efficacious, and at the same time
relatively safe and well tolerated. Chemo-enzymatic peptide synthesis (CEPS) using peptide ligases
features excellent purity and yield, thus becoming an attractive method to replace traditional chemical
synthesis method for synthesizing peptide drugs. However, the development of efficient and versatile
peptide ligases lags behind, limiting the advancement of peptide therapeutics. Fundamentally, this is
due to the inefficiency of current peptide ligase engineering method that relies on rational protein
engineering coupled with low throughput enzymatic characterization. We will overcome this limitation
by developing a specialized microfluidic system for high throughput peptide ligase engineering. We
will merge modern biochemistry and molecular biology methods with advanced droplet microfluidics
to enable high throughput screening of peptide ligase variants.
In this project, we will build an enzyme screening platform and demonstrate its capacity on increasing
aminolysis to hydrolysis ratio of subtiligase (a well characterized peptide ligase). In Specific Aim 1,
we will develop a microfluidic system for ultrahigh-throughput and quantitative analysis of
subtiligases. We will develop the microfluidic hardware, processes, and assays to enable the analysis
and screening of a large number (over 106) of variants of subtiligase. In Specific Aim 2, we will
establish and test subtiligase screening platform. We will design and synthesize subtiligase variant
library and we will demonstrate the throughput and sensitivity of our microfluidic system using a mock
subtiligase variant library. We will also establish kinetic assays for charactering subtiligase. Achieving
these aims will prove that peptide ligase assay with high sensitivity can be incorporated with droplet
microfluidic components to enable high throughput engineering of peptide ligase. A proposed phase II
project would involve screening of subtiligase libraries to increase aminolysis to hydrolysis ratio of
subtiligase and applying our engineering system to engineer other important properties of peptide
ligases such as substrate selectivity and racemization activity as well as early production of hardware
and disposables.PROJECT NARRATIVE
During the past decade, therapeutic peptide research is experiencing a renaissance with more than 60 FDA-
approved peptide medicines in market and an estimated US$25 billion market value, however, the traditional
manufacturing process of peptides (especially those longer ones) is cumbersome to fulfill the ever-increasing
demand of peptide therapeutics. Chemo-enzymatic peptide synthesis (CEPS) features excellent purity and yield,
but is limited by the availability of efficient and versatile peptide ligases, the key enzyme for performing
ligation reaction between peptide to peptide to synthesize longer peptides. We will develop a high throughput
screening platform specializing on engineering peptide ligases to improve their efficiency through merging
modern biochemistry, molecular biology with droplet microfluidics.

* Information listed above is at the time of submission. *

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