Textbook on Cloning, Expression and Purification of Recombinant Proteins

This book is immensely useful for graduate students as well as researchers to understand the basics of molecular biology and Recombinant DNA Technology. It provides a comprehensive overview of different approaches for the synthesis of recombinant proteins from E. coli including their cloning, expression and purification. Recent advances in genomics, proteomics, and bioinformatics have facilitated the use of Recombinant DNA Technology for evaluating the biophysical and biochemical properties of various proteins. The book starts with an introductory chapter on gene cloning, protein expression and purification and its implication in current research and commercial applications. Each chapter provides a lucid set of principles, tools and techniques for both students and instructors. The protocols described have been aptly exemplified, and troubleshooting techniques have been included to aid better understanding. Moreover, the set of questions at the end of each chapter havebeen particularly formulated to help effective learning.

Auteur

Dr. Kakoli Bose is currently working as Scientific Officer 'G' at Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India. She is known for her expertise in a diverse range of fields from Protein Biochemistry, Enzymology, Structural Biology, Biophysics to Cell Biology and Computational Biology. Her research activities are focused on elucidation of the role of macromolecules that are involved in pathways of apoptosis and their associated diseases. She is an active member of many elite scientific societies and associations, including Biochemical Society, UK, Indian Biophysical Society, Indian Association for Cancer Research and American Chemical Society. Dr. Bose is also a core member of the Editorial Board of coveted international journals such as Bioscience Reports and the Biochemical Journal, Portland Press, UK. She is a recipient of many prestigious awards, including the National Women Bioscientist Award from Govt. of India. She has a teaching experience of more than 10 years in the fields of Biochemistry, Biophysics, and Structural Biology. She has several publications in reputed international journals to her credit. Furthermore, her publication involving a four-state equilibrium unfolding process remains a landmark and pioneering work till date.

Contenu

Module 1_Introduction.- Chapter 1. History of recombinant DNA technology.- Chapter 2. Overview of technology.- Chapter 3. It's impact on research.- Module 2_Cloning basics.- Chapter 3. c-DNA library.- Chapter 4. PCR amplification.- Chapter 5. Restriction Digestion.- Chapter 6. Ligation.- Chapter 7. Colony screening.- Chapter 8. Transformation.- Chapter 9. Trouble-shooting.- Chapter 10. Problems.- Module 3_Choice of vectors (expression and cloning).- Chapter 11. Different types of vectors.- Chapter 12. Promoters.- Chapter 13. Antibiotic selection.- Chapter 14. Sequence confirmation.- Chapter 15. Trouble-shooting.- Chapter 16. Problems.- Module 4_Transformation and protein expression.- Chapter 17. Competent cell preparation.- Chapter 18. Transformation methods.- Chapter 19. Different bacterial strains (E. coli, Pseudomonas, Streptomyces).- Chapter 20. Optimizing protein expression (different inducers, temperature, concentration of inducers).- Chapter 21. Expression in insect cells.- Chapter 22. Trouble-shooting.- Chapter 23. Problem. Module 5_Protein purification.- Chapter 24. Lysis and extraction.- Chapter 25. Checking solubility and designing purification strategies.- Chapter 26. Databases and tools to determine physico-chemical properties of protein.- Chapter 27. Salting out and salting in methods.- Chapter 28. Dealing with proteins in inclusion bodies.- Chapter 29. Choice of buffer and dialysis.- Chapter 30. Overview of chromatography.- Chapter 31. Choice of chromatographic techniques with specific examples.- Chapter 32. Trouble-shooting.- Chapter 33. Problems.- Module 6_Affinity chromatography.- Chapter 34. Overview.- Chapter 35. His6.- Chapter 36. Cobalt.- Chapter 37. Streptavidin.- Chapter 38. MBP.- Chapter 39. GST.- Chapter 40. Trouble-shooting.- Chapter 41. Problems.- Module 7_Ion exchange chromatography.- Chapter 42. Overview.- Chapter 43. Cation exchange.- Chapter 44. Anion exchange.- Chapter 45. Choice of different combination of chromatographic techniques with examples.- Chapter 46. Trouble-shooting.- Chapter 47. Problems.- Module 8_Gel filtration chromatography.- Chapter 48. Overview.- Chapter 49. Different columns.- Chapter 50. HPLC.- Chapter 51. FPLC.- Chapter 52. Trouble-shooting.- Chapter 53. Problems.- Module 9_Purification of difficult proteins.- Chapter 54. Insoluble protein purification.- Chapter 55. Toxic protein purification.- Chapter 56. Membrane protein purification.- Chapter 57. Trouble-shooting.- Chapter 58. Problems .- Module 10_Quantitation and characterization.- Chapter 59. Need for protein quantitation.- Chapter 60. Different methods (Bicinchoninic Acid (BCA), Bradford, Folin-Lowry, Kjeldahl, UV absorption).- Chapter 61. Calculation of protein concentration.- Chapter 62. Purity calculation.- Chapter 63. Trouble-shooting.- Chapter 64. Problems. Module 11_Characterizing recombinant proteins.- Chapter 65. Secondary structure (CD).- Chapter 66. Tertiary structure (CD, fluorescence).- Chapter 67. Functional studies (protease, kinase, phosphatase etc).- Chapter 68. Preliminary Biochemical assays.- Chapter 69. Trouble-shooting.- Chapter 70. Problems.