Contents & References of Expression and purification of recombinant FGFR2b protein and investigation of its structural changes due to interaction with toxic metals
List:
Chapter One Introduction and importance of the topic. 3
1-1- Introduction. 4
1-1-1- fibroblast growth factor. 4
1-1-2- fibroblast growth factor receptors. 4
1-1-3- type 2 fibroblast growth factor receptors. 5
1-1-4- fibroblast growth factor tyrosine kinase receptors. 6
1-1-5- activation of kinases through mutation. 8
1-2- The importance of the topic and the necessity of conducting research. 9
1-3- Plan objectives. 9
1-3-1- the main goal. 9
1-3-2- Secondary objectives. 9
1-3-3- practical goals. 10
1-3-4- Assumptions. 10
1-3-5- Variables. 10
The second chapter Review of past texts. 11
2-1- protein. 12
2-1-1- The structure of proteins. 13
2-1-2-tyrosine kinase receptors. 17
2-1-3- fibroblastic growth factors. 18
2-1-4- fibroblast growth factor receptors. 20
2-1-5- fibroblast growth factor receptors and pathological disorders. 22
2-1-6- cellular signaling pathway of fibroblast growth factors. 25
2-1-7- Regulation of fibroblast growth factor signaling pathway. 29
2-2- What is metal?. 31
2-2-1- Toxic metals. 31
2-2-3- Lead. 32
2-2-4-Cadmium. 32
2-2-5- Nickel. 33
2-2-6-aluminum. 33
2-7- The effect of metals on signaling pathways. 34
2-7-1-ROS. 34
2-7-2-MAPK. 35
2-7-3-PI3K/Akt 36
2-7-4-HIF. 37
2-7-5- NF-?B. 38
2-7-6-NFAT. 39
2-7-7- AP. 40
2-8- Effect of metal compounds on signaling pathways and gene expression. 41
2-8-1- lead. 41
2-8-2- Cadmium. 43
2-8-3- Nickel. 46
2-8-4- Aluminum. 48
2-9- Predicting the structure of proteins. 50
2-9-1- Examining the structure of proteins. 50
2-9-2- Structural study of proteins. 51
2-9-3- Techniques for studying the structure of proteins. 52
2-9-4- spectroscopic fluorescence technique. 53
2-9-5- Circular dilation technique (CD) 56
2-9-6- Denaturation techniques and agents. 57
2-10- Production and purification techniques of recombinant proteins. 60
2-10-1- Use of recombinant proteins. 60
2-10-2- Recombinant proteins 60
2-10-3- Production of recombinant proteins in plants. 63
2-10-4- Using Escherichia coli as a host organism for protein production 64
2-10-5- Purification of recombinant proteins. 64
Chapter 3 materials and methods. 66
3-1- Test materials, equipment and variables. 67
3-1-1- Materials used in the experiment. 67
3-1-2- Devices and equipment used in testing. 68
3-2- Solutions and buffers. 69
3-2-1- Preparation of ampicillin stock. 69
3-2-2- Preparation of IPTG stock. 70
3-2-3- Preparation of cell lysing buffer to check protein solubility. 70
3-2-4- preparation of cell lysing buffer for protein purification. 70
3-2-5- Preparation of 4% polyacrylamide gel solution. 70
3-2-6- Preparation of 12% polyacrylamide gel solution. 71
3-2-7- Preparation of 10% APS solution. 71
3-2-8- Preparation of x10 electrode buffer (Running buffer) 71
3-2-9- Preparation of Sample Buffer) 71
3-2-10- Preparation of 0.5 M Tris-Hcl solution. 72
3-2-11- Preparation of 1.5 M Tris-Hcl solution. 72
3-2-12- Preparation of Staining Buffer. 72
3-2-13- Preparation of Destaining Buffer. 73
3-2-14- Preparation of acrylamide-bisacrylamide solution. 73
3-2-15- Preparation of SDS buffer. 73
3-2-16- Preparation of buffer A (Washing Buffer) 73
3-2-17- Preparation of buffer B (Eluting Buffer. 74
3-2-18- Preparation of dialysis buffer. 74
3-2-19- Preparation of guanidine hydrochloride stock (GnHCl) 74
3-2- Preparation of metal stocks 74 3-3 Preparation of recombinant plasmid pLEICS-01 and recombinant protein tyrosine sequence 75
3-2- Recombinant protein production steps
3-3- Determination of protein concentration
3-3-Spectroscopic studies83
3-3-4- Fluorescence spectroscopic studies. 83
3-3-5- CD ring imaging spectroscopic studies) 85
3-3-6- FTIR spectroscopic studies. 86
3-3-7-Chemical denaturation studies using fluorescence spectroscopy 87
Chapter 4 findings and results. . 88
4-1- Examining protein expression at 37°C. 88
4-2- Examination of protein expression at 20 degrees Celsius. 89
4-3- Investigating the solubility of expressed protein at two temperatures of 20 and 37 degrees Celsius. 90
4-4- Checking the purity of dissolved protein. 91
4-5- SDS-PAGE analysis after dialysis. 92
4-6- Investigating the function of purified protein. 93
4-7- Investigating the tertiary structure of the tyrosine kinase region of the recombinant FGFR2b protein 94
4-8- Investigating the effect of chemical denaturation on the maximum wavelength of the intrinsic fluorescence emission of the tyrosine kinase region of the recombinant FGFR2b protein 98
4-9- Investigating the effect of chemical denaturation on the intensity of the intrinsic fluorescence emission of the protein tyrosine kinase region Recombinant FGFR2b 101
4-10- Investigating the effect of lead, cadmium, aluminum and nickel on the second structure of the tyrosine kinase region of recombinant FGFR2b protein 104
Chapter 5 Discussion and conclusion. 109
5-1- Discussion and conclusion. 110
List of references: 116
.Abstract
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