A Caltech Library Service

Improvement of Integral Membrane Protein Expression via Optimization of Simulated Integration Efficiency


Marshall, Stephen Sandell (2018) Improvement of Integral Membrane Protein Expression via Optimization of Simulated Integration Efficiency. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9Q23XD3.


Integral membrane protein characterization is limited by the low levels of protein obtainable from heterologous overexpression in hosts such as Escherichia coli. Differences in the efficiencies of subdomains of the co-translational integration processes of membrane proteins into the membrane could explain the observed variation in the experimental expression of closely related homologs in E. coli. We have developed a method to predict and increase the expression of individual membrane proteins by optimizing the efficiency of their translocon-mediated integration into the membrane. The integration efficiency of each component of a membrane protein is calculated using a coarse-grained co-translational simulated integration model. The results of model simulations, experimental expression levels quantified by integral membrane protein-GFP fusion fluorescence, and a novel antibiotic survival test that reports on misintegration in vivo are applied to test the relationship between the integration efficiency of specific domains and experimental expression. Changes in simulated integration efficiencies due to sequence modifications agree with the effects on experimental expression in vivo. In the case of the TatC protein family, misintegration of the C-tail is found to be a major contributor to expression failure in E. coli. Beneficial sequence modifications that improve both simulated integration efficiency and experimental expression levels can be identified using the model. Preliminary evidence shows that simulated integration efficiency could potentially predict the effects of mutations on Haemophilus influenzae GlpG experimental expression in E. coli. The process described herein allows for the rational overexpression of integral membrane proteins through the identification and mitigation of inefficiencies in the underlying co-translational membrane integration process.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:membrane protein expression
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Clemons, William M.
Thesis Committee:
  • Shan, Shu-ou (chair)
  • Mayo, Stephen L.
  • Clemons, William M.
  • Miller, Thomas F.
Defense Date:28 August 2017
Record Number:CaltechTHESIS:08292017-151537318
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Ch. 2
Marshall, Stephen Sandell0000-0003-2263-6854
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10397
Deposited By: Stephen Marshall
Deposited On:11 Sep 2017 20:58
Last Modified:04 Oct 2019 00:17

Thesis Files

PDF - Final Version
See Usage Policy.


Repository Staff Only: item control page