Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Matthew D. Smith

Advisor Role

Thesis Supervisor

Abstract

Arabidopsis thaliana protein Tic20 (atTic20) is a member of the translocon at the inner envelope membrane of chloroplasts. Evidence to date suggests it is part of the main preprotein conducting aperture in the complex, but its exact role is still debated. To help characterize its role, a protocol optimizing yield and purity of recombinantly expressed atTic20 was developed, and a series of experiments was performed to examine its secondary structure and its ability to interact with chloroplast transit peptides. The attempt to increase protein yield was successful, with growth at 20oC in the auto-inducing media ZYP-5052 showing the greatest potential for recombinant protein expression. Interestingly, expression under these conditions resulted in the targeting of recombinant atTic20 to the bacterial membrane, which necessitated the adoption of a membrane isolation and solubilization procedure. This resulted in an almost eight-fold increase in protein yield per litre of culture compared to an inclusion body extraction procedure. Circular dichroism showed that atTic20 has largely α-helical conformation in liposomes, with a short, intrinsically disordered domain at its N-terminus. With regards to transit peptide interaction, solid phase binding assays and circular dichroism spectroscopy of atTic20 and putative binding partners indicated that atTic20 does not recognize chloroplastic targeting sequences. This strengthens the case for atTic20 as a primarily structural protein.

Convocation Year

2014

Download

Share

COinS