Functional and Structural Basis of PPM1K Catalytic Mechanism

声明

Abstract

摘要

Table of contents

List of tables

List of figures

List of acronyms

Chapter 1-General introduction

1．3．1 Maple syrup urine disease

1．3．2 BCKAD complex

1．3．3 From kinase to phosphatase

1．3．4 Phosphorylation mechanism history

1．3．5．Branched Chain Amino acids Role in CNS

1．3．6 Presence of the Third Metal Ion in PP2C Family

1．4 Summary

Chapter 2-Vector Construct

2．1 Construct

2．1．2 BDP DNA insert

2．1．3 Protein Tags

2．2 PCR

2．2．1 Introduction

2．2．2 Site-Directed Mutagenesis(Quick-Change)

2．2．3-SLIC(sequence-and ligation-independent cloning)Method

2-3 Result

2．6 Conclusion

Chapter 3-Expression and Protein purification

3．1 Introduction

3．1．1 Affinity chromatography

3．1．2 Gel chromatography

3．1．3 Ion-Exchange

3．2 Material and methods

3．2．1．Materials

3．2．2 Methods

3．3 Results

3．3．1 Protein purification of BDP(30-372)by Tris buffer pH 7．5

3．4 Conclusion and discussion

Chapter 4-Enzyme Assays

4．1．1 Enzyme as biocatalyst

4．1．2．Enzyme inhibition

4．1．3 IC50

4．2 Material and methods

4．3 Results

4．4 Conclusion and discussion

Chapter 5-Protein crystallization

5．1．2-Crystallization of biological macromolecules

5．1．3．Macromolecular crystallization

5．1．4．Crystal structure determination

5．2 Material and methods

5．3 Results

5．4 Conclusion

5．5 Discussion

References

Appendices