Cloning, Expression, and Characterization of a HEAT Repeat Solenoid Protein Público

Dhaliwal, Olivia Mangat (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/td96k284s?locale=pt-BR
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Abstract

A HEAT-like solenoid protein was chosen for this project due to published evidence of its thermodynamic stability and flexibility in sequence and structure. Two DNA sequences were designed to clone varying lengths of a repeated HEAT-like protein for expression in E. coli, without N- and C- cap repeated units to permit self-assembly. Expressed proteins were purified in an inclusion-body washing protocol and affinity column purification procedures. Purified proteins were assayed for thermodynamic stability, secondary structure, and homogeneity in assemblies. Proteins that had undergone a refolding protocol in a series of refolding buffers were compared to proteins that had been only dialyzed in deionized water. Refolded proteins had more intense and consistent alpha-helical CD signatures and displayed longer, more ordered tubular assemblies when imaged using TEM. Non-refolded proteins had noisy, inconsistent CD signatures and their assemblies were fragmented and disordered.

Table of Contents

Introduction 1

Literature Review 3

Materials and Methods 11

Sequence design, cloning, and expression 12

Lysis, inclusion body washing, and solubilization 16

Affinity column purification 18

Refolding and assembly 19

Characterization 20

Results and Discussion 21

Non-Refolded Proteins 23

Refolded Proteins 26

Conclusion and Future Aims 30

Supplemental Information 32

References 36

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