Engineering spores to display g protein-coupled receptors for directed evolution

All human cells are surrounded by a plasma membrane made from a phospholipid bilayer, which is responsible for maintaining a biologically active species, while stopping entry of deleterious substances from the outside. G protein-coupled receptors (GPCRs) are the membrane proteins, which transmit signals across the cell membrane. GPCRs are involved in almost every physiological process, and irregular control leads to pathological conditions. Therefore, they are major drug targets. Crystal structure determination is required to understand the molecular details of activation/deactivation. However, GPCRs are difficult to crystallize because of stability issues. An efficient protein engineering system needs to be developed. The goal is to design and create a system to display a heterologous protein on the Bacillus subtilis spore coat. Human parathyroid hormone receptor (huPTH1R) is used as a model system. HuPTH1R is a GPCR, which is vital in regulating calcium and phosphate levels in the blood. Molecular biology is used to create the plasmid pDG1730 huPTH1R-CotC that fused huPTH 1 R to a spore coat protein, CotC. The pl asmid is transformed into B. subtilis, and huPTH1 R is successfully integrated into B. subtilis genome via recombination. This work represents the first system for GPCR display on the spore coat. Spore display overcomes many of the hurdles found in "traditional" protein display systems. Finally, this system can be used as a general method for engineering and optimizing membrane proteins by directed evolution.

Name thesis:degree_name

Master of Science in Pharmaceutical Engineering - (M.S.)

Discipline thesis:degree_discipline

Chemical, Biological and Pharmaceutical Engineering

Year

2013

Author dc:creator

• Kim, Alyssa Misoo

Contributors dc:contributor

• Edgardo Tabion Farinas
• Piero M. Armenante
• Laurent Simon

• Protein engineering
• G protein-coupled receptors
• Chemical Engineering
• Pharmaceutics and Drug Design

Repository record dc:identifier

https://digitalcommons.njit.edu/theses/174

OAI identifier oai:identifier

oai:digitalcommons.njit.edu:theses-1173