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Practical Guide to Protein Engineering 1st ed. 2020 [Mīkstie vāki]

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  • Formāts: Paperback / softback, 202 pages, height x width: 240x168 mm, weight: 454 g, 68 Illustrations, color; 23 Illustrations, black and white; XV, 202 p. 91 illus., 68 illus. in color., 1 Paperback / softback
  • Sērija : Learning Materials in Biosciences
  • Izdošanas datums: 30-Oct-2020
  • Izdevniecība: Springer Nature Switzerland AG
  • ISBN-10: 3030568970
  • ISBN-13: 9783030568979
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Practical Guide to Protein Engineering 1st ed. 2020Palielināt
  • Formāts: Paperback / softback, 202 pages, height x width: 240x168 mm, weight: 454 g, 68 Illustrations, color; 23 Illustrations, black and white; XV, 202 p. 91 illus., 68 illus. in color., 1 Paperback / softback
  • Sērija : Learning Materials in Biosciences
  • Izdošanas datums: 30-Oct-2020
  • Izdevniecība: Springer Nature Switzerland AG
  • ISBN-10: 3030568970
  • ISBN-13: 9783030568979
Citas grāmatas par šo tēmu:
Permanent link: https://www.kriso.lv/db/9783030568979.html
Keywords:

This textbook introduces readers in an accessible and engaging way to the nuts and bolts of protein expression and engineering. Various case studies illustrate each step from the early sequence searches in online databases over plasmid design and molecular cloning techniques to protein purification and characterization. Furthermore, readers are provided with practical tips to successfully pursue a career as a protein engineer. With protein engineering being a fundamental technique in almost all molecular biology labs, the book targets advanced undergraduates and graduate students working in molecular biology, biotechnology and related scientific fields.


1 Literature Search
1(10)
1.1 Databases
2(1)
1.2 How to Search?
3(2)
1.3 Important Information
5(6)
Exercise
10(1)
Further Reading
10(1)
2 Sequence Analysis
11(18)
2.1 Gene Information
12(5)
2.1.1 Kyoto Encyclopedia of Genes and Genomes (KEGG)
13(1)
2.1.2 National Center for Biotechnology Information (NCBI)
14(1)
2.1.3 SnapGene
15(1)
2.1.4 Other Databases of Protein Sequences
15(2)
2.2 Gene Analysis
17(12)
2.2.1 Protein Sequence Analysis
19(5)
2.2.2 DNA Sequence Analysis
24(2)
2.2.3 Perl
26(1)
Exercise
27(1)
Further Reading
27(2)
3 Structural Analysis
29(10)
3.1 Protein Structure Databases
30(2)
3.1.1 Protein Data Bank (PDB)
30(2)
3.1.2 Biological Magnetic Resonance Data Bank (BMRB)
32(1)
3.2 Protein Structure Prediction
32(5)
3.2.1 Protein Secondary Structure Prediction
33(1)
3.2.2 Protein Tertiary Structure Prediction
33(4)
3.3 Protein Structure Visualization Tools
37(2)
Exercise
38(1)
Further Reading
38(1)
4 Protein Expression Hosts and Expression Plasmids
39(24)
4.1 Escherichia coli Strains
40(1)
4.2 Plasmid and Plasmid Map
41(18)
4.2.1 Finding a Plasmid
41(4)
4.2.2 Reading a Plasmid Map
45(1)
4.2.3 Creating a Plasmid Map
45(1)
4.2.4 Plasmid Selection
46(13)
4.3 Choosing an Appropriate Host
59(4)
Exercise
59(2)
Further Reading
61(2)
5 Gene Cloning
63(24)
5.1 Gene Amplification from Genomic DNA
65(7)
5.1.1 Ordering a Bacterial Strain or Its Genomic DNA
65(1)
5.1.2 Bacterial Cultivation and Genomic DNA Extraction
66(2)
5.1.3 Gene Amplification
68(4)
5.2 Gene Synthesis
72(3)
5.2.1 Gene Design
73(1)
5.2.2 Ordering a Synthetic Gene and Codon Optimization
74(1)
5.3 Molecular Cloning
75(7)
5.3.1 Restriction and Ligation
76(2)
5.3.2 NEBuilder® HiFi DNA Assembly
78(3)
5.3.3 PTO-QuickStep
81(1)
5.4 Clone Verification
82(5)
Exercise
85(1)
Further Reading
86(1)
6 Protein Expression
87(6)
6.1 Protein Expression Medium
89(2)
6.2 Protein Expression Conditions
91(1)
6.3 Inclusion Body
91(2)
Exercise
92(1)
Further Reading
92(1)
7 Assay
93(28)
7.1 Assay Detection Mode and Formats
94(16)
7.1.1 Assay Detection Mode
94(9)
7.1.2 Assay Formats
103(7)
7.2 Key Considerations in Assay Development
110(5)
7.2.1 Assay Conditions
110(3)
7.2.2 Understanding Your Assay
113(2)
7.2.3 Information You Can Derive from an Assay
115(1)
7.3 Selection Versus Screening
115(6)
Exercise
118(1)
Further Reading
119(2)
8 Gene Mutagenesis
121(28)
8.1 Nucleotide Substitution and Amino Acid Substitution
122(3)
8.1.1 Nucleotide Substitution
122(2)
8.1.2 Amino Acid Substitution
124(1)
8.2 Organization of the Genetic Code
125(3)
8.3 Protein Engineering Approaches
128(4)
8.3.1 Directed Evolution
131(1)
8.4 Mutagenesis
132(17)
8.4.1 Random Mutagenesis
133(6)
8.4.2 Focused Mutagenesis
139(8)
Exercise
147(1)
Further Reading
147(2)
9 High-Throughput Screening (HTS)
149(20)
9.1 Practical Aspects of HTS
150(6)
9.1.1 Liquid Handling
150(1)
9.1.2 Internal Control
151(1)
9.1.3 High-Throughput Cultivation
152(2)
9.1.4 Cell Disruption
154(1)
9.1.5 Spectroscopic Measurement
155(1)
9.2 Assay Conditions for Screening
156(4)
9.2.1 Substrate Concentration
157(1)
9.2.2 Reaction Time
158(1)
9.2.3 Enzyme Concentration
159(1)
9.2.4 Reaction Termination
159(1)
9.3 How Good Is Your HTS System?
160(3)
9.3.1 Background Plate Versus Wildtype Plate
160(1)
9.3.2 Assessment of HTS Data
160(3)
9.4 Analysing and Presenting Library Screening Result
163(2)
9.4.1 Internal Controls
163(1)
9.4.2 Data Presentation
164(1)
9.5 Re-screening
165(1)
9.6 Adjusting Your HTS System as You Go Along
165(4)
Exercise
167(1)
Further Reading
168(1)
10 Protein Variants Analysis and Characterization
169(18)
10.1 Mutation
170(3)
10.1.1 DNA Sequencing and Sequencing Chromatogram
170(2)
10.1.2 Structure-Function Relationships
172(1)
10.1.3 Additive Interaction Versus Epistatic Interaction
173(1)
10.2 Protein Purification
173(5)
10.2.1 Protein Purification Methods
174(1)
10.2.2 Protein Purification Strategy
175(2)
10.2.3 Protein Quantification
177(1)
10.3 Protein Characterization
178(9)
10.3.1 Progress Curve for Enzyme-Catalysed Reaction
179(5)
10.3.2 Enzyme Inhibition
184(1)
Exercise
185(1)
Further Reading
186(1)
11 Continuing from Protein Variants
187(10)
11.1 DNA Recombination
188(3)
11.1.1 Staggered Extension Process (StEP)
189(2)
11.1.2 Diluting Mutations Using DNA Recombination Methods
191(1)
11.2 Saturation Mutagenesis
191(6)
11.2.1 Randomization Scheme
192(3)
11.2.2 Designing a Saturation Mutagenesis Experiment
195(1)
Exercise
195(1)
Further Reading
195(2)
12 Employability
197
12.1 Sectors Requiring Protein Engineering Expertise
199(1)
12.2 What Are the Skills Employers Are Looking for?
199(1)
12.3 How to Improve Your Curriculum Vitae (CV)?
200(1)
12.4 Career Planning
200(1)
12.5 Where to Look for Career Resources?
201(1)
12.6 Where to Look for a Job?
201
Exercise
202(1)
Further Reading
202
Dr. Tuck Seng Wong is a Senior Lecturer (Associate Professor) from the Department of Chemical & Biological Engineering at the University of Sheffield (UK). He is also a Visiting Professor at the National Center for Genetic Engineering and Biotechnology (BIOTEC) in Thailand. Dr. Wong leads a Biocatalysis & Synthetic Biology group in Sheffield, and his research focuses on sustainable biomanufacturing using engineered enzymes or microbes. His passion in engineering of biology is inspired by his research supervisors and mentors including Nobel Laureate Prof. Frances H. Arnold (Caltech), Prof. Ulrich Schwaneberg (Bremen), Prof. Sir Alan R. Fersht (Cambridge) and Prof. Alexander Steinbüchel (Münster). Dr. Wong obtained his BEng in Chemical Engineering (1st class honour) from the National University of Singapore, followed by an MSc and a PhD (special distinction, highest possible grade) in Biochemical Engineering from the Jacobs University Bremen in Germany. He is a recipient of multiple prestigious fellowships, including the RAEng|The Leverhulme Trust Senior Research Fellowship (2019), the Royal Academy of Engineering Industrial Fellowship (2016) and the MRC Career Development Fellowship (2007 2009). He was one of the 10 young academics appointed to the EPSRC Early Career Forum in Manufacturing Research in 2014 and a world finalist of the Synthetic Biology Leadership Excellence Accelerator Program (LEAP) in 2015. He has published over 40 papers, mostly in the areas of protein engineering and synthetic biology.











Dr. Kang Lan Tee is a Lecturer (Assistant Professor) from the Department of Chemical & Biological Engineering (CBE) at the University of Sheffield (UK). She has published over 20 papers in the field of protein engineering and synthetic biology since 2004. Dr. Tee is also the co-Founder of protein engineering start-up SeSaM-Biotech GmbH and a Global Challenges Research Fellow (2019 2021). Her research team applies protein engineering strategies to address global challenges in biomanufacturing and sustainability. She also develops and leads the biorefineries module in CBE