Developing a 2-Phase Screening System to Assess the in vitro and in vivo Stability of Engineering Native Seed Storage Proteins

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Creator: 

Jaworski, Allison

Date: 

2015

Abstract: 

Seed storage proteins are the nitrogen and sulfur reserves for the germinating embryo and comprise the majority of the protein content of a seed (Shewry et al, 1995; Tandang-Silvas et al, 2010). The 11S globulins are the principal seed storage proteins in members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties (e.g. emulsification and gelling) and nutritional value, with respect to amino acid content, of their seed protein extracts (Muntz et al, 1998; Tandang-Silvas et al, 2011). The factor that has limited the success of these studies to date is insufficient accumulation of the engineered variants in vivo due to their reduced stability, compared to the native protein. To address this issue, this thesis describes the development of a 2-phase screening system that facilitates protein engineering of native 11S globulins by assessing the ability of stable site-directed variants to accumulate in seeds. Phase I employs E. coli as an expression system to assess the thermodynamic stability of the engineered seed storage proteins in vitro. Methionine-enriched 11S globulins with stability parameters similar to the wild-type protein are then expressed in A. thaliana in phase II, the in vivo component of the screen, to assess the ability of the engineered protein to accumulate in plant seeds. To complement the novel 2-phase screening system, a comprehensive survey of 67-methionine-enriched variants of a model A. thaliana 11S globulin was completed, employing fluorescence-monitored guanidine hydrochloride denaturation to identify regions of the protein that are amenable to the introduction of methionine residues. This survey demonstrates that methionine can be introduced into 11S globulins but single locations cannot be treated as sinks for the introduction of multiple, sequential methionine residues. The use of HotSpot Wizard in conjunction with available structural data is a successful strategy for short-listing residues as targets for substitution to methionine. This thesis developed tools, a 2-phase screen and an information platform identifying regions within the 11S globulin structure that are accepting of methionine introduction, to facilitate subsequent protein engineering studies attempting to alter the physicochemical or nutritional properties of seed proteins.

Subject: 

Food Science and Technology
Biology - Molecular

Language: 

English

Publisher: 

Carleton University

Contributor: 

Supervisor: 
Aitken, Susan M.

Thesis Degree Name: 

Doctor of Philosophy: 
Ph.D.

Thesis Degree Level: 

Doctoral

Thesis Degree Discipline: 

Biology

Parent Collection: 

Theses and Dissertations

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