Cloning, Expression, and Characterization of a HEAT Repeat Solenoid Protein Public
Dhaliwal, Olivia Mangat (2016)
Published
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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Cloning, Expression, and Characterization of a HEAT Repeat Solenoid Protein () | 2018-08-28 12:20:30 -0400 |
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