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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.


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CaltechTHESIS  

Autor(es)

Marshall, Stephen Sandell - 

Id.: 70082640

Versión: 1.0

Estado: Final

Tipo:  application/pdf - 

Tipo de recurso: Thesis  -  NonPeerReviewed  - 

Tipo de Interactividad: Expositivo

Nivel de Interactividad: muy bajo

Audiencia: Estudiante  -  Profesor  -  Autor  - 

Estructura: Atomic

Coste: no

Copyright: sí

Formatos:  application/pdf - 

Requerimientos técnicos:  Browser: Any - 

Relación: [References] http://resolver.caltech.edu/CaltechTHESIS:08292017-151537318
[References] http://thesis.library.caltech.edu/10397/

Fecha de contribución: 19-sep-2017

Contacto:

Localización:
* 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. http://resolver.caltech.edu/CaltechTHESIS:08292017-151537318

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