Purifying and Characterising a Sco1-Like Protein 3966 in Streptomyces Lividans

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ACKNOWLEDGEMENT

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DECLARATION

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ABSTRACT

In this study, Protein purification is a series of processes to isolate a single type of protein in a complex mixture. The purification of proteins is vital for the characterization of the function, structure of the protein interactions of interest. The starting material is usually a biological tissue culture or microbial. There are several steps in the process of purification can release the protein matrix that confines it, separate the protein and protein parts of the mixture, and finally separate the desired protein from all others. This last step may be the most laborious protein purification.

This study is a technology advancement paving the way for the usage of top-down mass spectrometry for characterization of other large integral membrane proteins.

TABLE OF CONTENT

ACKNOWLEDGEMENTii

DECLARATIONiii

ABSTRACTiv

TABLE OF CONTENTv

CHAPTER NO 1: INTRODUCTION1

Background of study1

Rationale1

Problem Statement2

Aims & Objectives2

Significance2

Review of the Related Literature3

Protein Purification:4

Prolactin5

Streptomyces Lividans7

Physical Characteristics7

Molecular Characteristics7

Importance in Humans8

Synthesis of cytochrome c oxidase (sco) proteins:8

Molecular Characteristics8

Function of COX10

Isolation of Membrane Proteins11

Mechanism of action12

Other Functions13

Copper regulation13

Redox Signaling15

SCO in prokaryotes15

CHAPTER 2: MATERIALS AND METHODS19

Purification Techniques and Characterization of Proteins19

Material:20

Transformation:20

Restriction Digest:21

Over-Expression of 3966:22

Cell Lyses:23

Occurrence of Copper Proteins through the Three Domains of Life: A Bio-informatics Approach23

Ammonium Sulphate Precipitation:24

Dialysis of Protein:25

Column Chromatography:26

Q-Column:26

SP Column:26

Heparin column:26

G-75 Gel Filtration:27

SDS-PAGE:27

Cu (II) Titration:28

UV-visible Spectroscopy:28

Mass Spectrometry:29

Genome and Its Characteristics30

Transcriptome30

Proteome31

Metabolome33

Connectome33

Chromatin34

Q-SepharoseColumn42

SP-Column:44

Heparin Column:46

G-75 Gel Filtration Column Chromatography:48

Cu (II) Titration:51

CHAPTER NO 4: CONCLUSION57

Proteins can be purified by size, solubility, charge and binding affinity57

REFERENCES60

CHAPTER NO 1: INTRODUCTION

Background of study

Recent advancements in molecular biology have led to purification and characterization of proteins. This type of study is important, especially when molecular defects, such as lack of enzymes, or overproduction thereof, are associated with diseases that have seemed incurable in the past. Vaccines, gene therapy and replacement (such as that in insulin-deficiency) all have helped in improving health conditions, and have been based on good elucidation of structures, research on structure-function relationships, and establishment of protein purification specific to the amino acid sequence present.

With this in mind, this particular study designed a protocol to purify and characterize a synthesis of cytochrome oxidize (SCO)-1-like protein, identified in Streptomyceslividans and annotated P3966. As will be seen later, better understanding of SCO proteins is still warranted, as many potential functions of these types of proteins are unclear. Moreover, SCO is a vital protein in the assembly of cytochrome coxidase, and in essence the electron chain transport of the mitochondrial respiration mechanism, depends on it (Williams, 2005, 15202-15211).

Rationale

Initial studies of homologues in bacteria have been the usual first step in protein ...