This experiment is part of the iGEM 2019 Challenge Challenge Grant. Browse more projects

Stabilizing a mammalian protein by adding an isopeptide bond

$10
Pledged
1%
Funded
$2,345
Goal
17
Days Left
  • $10
    pledged
  • 1%
    funded
  • 17
    days left

About This Project

We are designing antibody domains stabilized by an internal isopeptide bond, an amide group between side chains. This structure is unusual for mammalian proteins, as the inside of proteins is hydrophobic. The motif is, however, common among certain prokaryotes. We will use a neural net to find residues to mutate to form isopeptide bonds. We hope if an internal isopeptide bond were inserted in a mammalian protein the half-life would be significantly greater, showing stabilizing.

Ask the Scientists

Join The Discussion

What is the context of this research?

Antibodies are frequently used in therapeutic contexts, as well as in assays during biochemical research. While they are relatively stable proteins, their half-life in the body (in the context of a biologic drug) could be extended if they weren't vulnerable to natural degradation mechanisms. We intend to insert an artificial bond into a single fragment of an antibody, as proof of concept that these proteins can be stabilized. This artificial 'isopeptide bond' is common to proteins extruded to the outside of some bacteria, but doesn't appear in mammalian proteins. This bond make the antibody less vulnerable to destruction in the body, which could create a longer lasting drug.

What is the significance of this project?

Antibody engineering has the potential to be an incredible tool both for pharmaceutical development and for biochemical tools such as 'enzyme linked immunosorbent assays.' The introduction of an isopeptide bond into a single domain of a mammalian antibody would be proof of concept for stabilization that would allow biologic drugs such as those used to treat autoimmune disease to last dramatically longer before being metabolized to inactive constituent parts. More stable antibodies also have the potential to create more robust assays for biochemical bench work, which would ultimately lower costs as the tests become less fragile.

What are the goals of the project?

This project will utilize a machine learning interface to identify the closest structural analogue to the antibody in question which already contains an isopeptide bond. Then we plan to use bioinformatic techniques to identify which residues must be altered to create a mutant antibody domain. Once these residues have been identified, we will express the mutated antibody fragments in E. coli, where they ought to remain soluble if they fold correctly. We will tag our antibodies with a fluorophore to see and quantify the soluble protein, which ought to contain the desired bond. We will test for the presence of this isopeptide bond within the folded antibodies using mass spectrometry.

Budget

Please wait...

We will be using the PCR kit to amplify our plasmids and then the restriction enzymes and site directed mutagenesis kit to alter them for isopeptide bond formation. We will express these plasmids in E coli cells to generate our proteins, and use the nickel chromatography column to purify our proteins. The plasmid miniprep kit will allow us to purify our plasmids and then send them off for sequencing to ensure that they have the mutations we intended to introduce. Conference registration is expensive, but will allow us to present our data to a group of peers in Boston next October!

Endorsed by

Morganne is truthfully one of the smartest people I know. If anyone deserves funding, it's someone like her with both the intelligence and the drive to make the absolute best use of it.

Flag iconProject Timeline

Our project begins with using computational techniques and previously published structural data to plan which antibody fragments will be synthesized in the lab.

From June 3rd, 20 antibody fragments from rational design will be made to get the desired isopeptide bond to form. We will try a further 50-150 DNA sequences from the machine learning.

By August 9th we will have finished our characterization, and in November we go to the iGEM conference!

As we go we will update this page and our wiki.

Jun 03, 2019

Start Lab Work

Jun 06, 2019

Project Launched

Jul 25, 2019

Finish our protein synthesis of antibodies

Aug 09, 2019

Finish characterization of antibodies to determine successful formation of isopeptide bond

Oct 15, 2019

Finish writing up our results and publishing them on the wiki (visit us at 2019.igem.org/Team:St_Andrews)

Meet the Team

Morganne Wilbourne
Morganne Wilbourne

Affiliates

University of St Andrews
View Profile
Eleonora Shantsila
Eleonora Shantsila
Thomas Storey
Thomas Storey

Affiliates

University of St Andrews
View Profile
Sarah Jackson
Sarah Jackson

Affiliates

University of St. Andrews
View Profile
Maggi Chalgunova
Maggi Chalgunova

Affiliates

University of St Andrews
View Profile
Zsolt Semperger
Zsolt Semperger

Affiliates

University of St Andrews
View Profile
Amy Buck
Amy Buck
Jack Briggs
Jack Briggs
Ben Gillen
Ben Gillen
Georgie Brown
Georgie Brown
Veronika Lachina
Veronika Lachina
James Hammond
James Hammond
Modelling Co-Leader

Affiliates

University of St Andrews
View Profile
Gregor Vagg
Gregor Vagg
Camille Young
Camille Young

Affiliates

University of St. Andrews
View Profile
Gavin Lamb
Gavin Lamb
Gordian Grüntuch
Gordian Grüntuch

Affiliates

University of St Andrews
View Profile

Team Bio

We're a group of undergraduates from the University of St Andrews competing in the 2019 iGEM competition. Our project revolves around synthesizing stable antibody fragments, and stems from our collective interest in biologic drug design. Some of us come from biological or chemical backgrounds, while others come from mathematical ones, but together we hope to be able to accomplish a lot this summer!

Morganne Wilbourne

Team Coordinator

Eleonora Shantsila

Modeling team co-leader and head of human practices

Thomas Storey

Modelling team member

Sarah Jackson

Modeling team member

Maggi Chalgunova

laboratory team member

Zsolt Semperger

laboratory team member

Amy Buck

Laboratory and Human Practices team member

Jack Briggs

modeling team member

Ben Gillen

modeling team member

Georgie Brown

laboratory team member

Veronika Lachina

modeling and laboratory team member

James Hammond

Modelling and lab member. Academic interests Systems and Developmental Biology. Recreational interests Chess, Hillwalking, and Lepidopterology.

Gregor Vagg

modeling team member

Camille Young

Laboratory team leader

Gavin Lamb

Modeling team member

Gordian Grüntuch

modeling and laboratory team member

Lab Notes

Nothing posted yet.


Project Backers

  • 1Backers
  • 1%Funded
  • $10Total Donations
  • $10.00Average Donation
Please wait...

See Your Scientific Impact

You can help a unique discovery by joining 1 other backers.
Fund This Project
Project journal