Class notes Protein engineering and recombinant proteins

Master in Advanced Molecular Sciences




PROTEIN ENGINEERING AND
RECOMBINANT PROTEINS




Chiara La Guidara
2021-2022
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, SUMMARY


DISCLAIMER: These lectures notes were written by AMS students older than you, but are not meant to be an official handbook
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PART I: THE LIFE OF A PROTEIN INSIDE CELLS 10

1. CELL STRUCTURE 10
1.1 Plasma membrane 10
1.2 Nucleus 10
1.3 Endoplasmic Reticulum (ER) 11
1.4 Golgi Apparatus 11
1.5 Mitochondria 11
1.6 Endosomes 11
1.7 Lysosomes 12

2. PROTEIN STRUCTURE 13
2.1 Introduction 13
2.1 Proteinogenic Amino acids 13
2.1.1 Chirality 13
2.1.2 Classification of AAs 14
2.1.3 Non-covalent interactions 16
2.2 The peptide bond 17
2.3 Levels of Protein structures 17
2.3.1 Primary structure and Protein Assembly 18
2.3.2 Secondary structure and Protein Folding 18
2.3.3 Tertiary structure and Packing of Proteins 21
2.3.4 Quaternary structure and Protein Interactions 22
2.4 Motifs, Domains, Folds, Families and Classes 22
2.5 Determining protein structure 23

3. PROTEIN BIOSYNTHESIS 26
3.3 Introduction 26
3.2 Codons 26
3.3 From RNA to protein: translation 26
3.4 Ribosomes in the cytoplasm of a eukaryotic cell 27
3.4.1 Composition of eukaryotic ribosomes 27
3.4.2 RNA-binding sites in the ribosome 28
3.5 tRNA molecules: matching amino acids codons in mRNA 28
3.6 Translation of the genetic code: two adaptors that act one after another; 29
3.6.1 mRNA translation mechanism and GTP consume 29
3.6.2 Initiation of the peptide chain 30
3.6.3 Elongation phase 31
3.6.4 Termination phase 31
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, 3.7 Prokaryotic vs eukaryotic mRNA molecules 32
3.8 Polyribosomes and Proteasomes 33
3.9 Mechanism of regulation of Protein Synthesis 34
3.9.1 The production of a protein by a eukaryotic cell 34
3.10.1 Protein import by membrane-bounded organelles. 35
3.10.2 The role of signal sequences in protein sorting 36
3.10.3 ER signal sequence and SRP: directing ribosomes to the ER membrane 36
3.10.4 Translocation of a soluble protein across the ER membrane 36
3.10.5 Integration of a transmembrane protein into the ER membrane 37
3.10.6 Integration of a double-pass transmembrane protein into the ER membrane 38
3.10.7 Vesicular traffic 38
3.10.8 Clathrin-coated pits and vesicles 38
3.10.9 Selective Transport: mediated by clathrin-coated vesicles 39
3.10.10 Model of transport vesicle docking 39
3.10.11 Transport vesicle fusion 40
3.10.12 Protein glycosylation in the ER 40
3.10.12 Exocytosis 41

4. NUTRIENT/DIETARY PROTEIN DEGRADATION 42
4.1 Introduction 42
4.2 Dietary Protein Is Enzymatically Degraded to Amino Acids 42
4.2.1 Removal of the amino group 42
4.2.2 The Urea cycle, the liver affair 43
4.3 Intracellular Protein Degradation 44
4.3.1 Consequence of impaired protein degradation 44
4.3.2 Protein degradation 45
4.4 The Ubiquitin/Proteasome System (UPS) 45
4.4.1 The role of Ubiquitin 45
4.4.2 Proteasomes 46
4.4.3 De-ubiquitination 47
4.5 Lysosomal degradation 48
4.5.1 The three pathways of autophagy 48
4.5.2 Macroautophagy 49
4.2.3 Basal autophagy 49
4.2.4 Selective Autophagy 49
4.2.5 Chaperone-mediated autophagy (CMA) 50
4.2.6 Autophagy receptors: molecular basis of autophagic selectivity 50
4.3 Ubiquitin-mediated protein degradation and lysosome degradation are connected 50

5. POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS 51
5.1 PTMs of proteins 51
5.2 Acetylation and Methylation 52
5.2.1 Acetylation 52
5.2.2 Acetylation on Histones 52
5.2.3 Methylation 53
5.2.4 Methylation VS Acetylation 54
5.2.5 Methylation, acetylation and phosphorylation of histones: the histone code. 54

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, 5.2.6 Acetylation of cytoplasmic proteins 55
5.3 Lipidation 56
5.3.1 Prenylation 57
5.3.2 Acylation 58
5.3.3 Differences between Myristylation and Prenylation 59
5.4 Phosphorylation 59
5.4.1 Kinases and phosphatases 60
5.4.2 SRC Kinase 61
5.5 Glycosylation 61
5.5.1 N-Glycosylation 61
5.5.2 O-Glycosylation 61
5.5.3 GPI Anchor 62
5.6 Redox Modifications 62
5.6.1 Disulphide bonds 62
5.6.7 Reactive Oxygen Species (ROS) 63
5.7 Limited proteolysis 64
5.7.1 RIP: Regulated intramembrane proteolysis 64

6. PROTEIN TARGETING 66
6.1 Cytosolic Pathway Vs Secretory Pathway 67
6.2 The Cytosolic Pathway 67
6.2.1 Protein Import And Export To Nucleus, Nls: Nuclear Localization Sequence 67
6.2.1 Protein Import To Mitochondria 70
6.2.3 Protein Import To Peroxisomes 70
6.2.4 Proteins Import To The Membrane, Membrane Localization Sequence 71
6.3 The secretory pathway 71

PART II: RECOMBINANT DNA AND HETEROLOGOUS PROTEINS EXPRESSION 72

7. MOLECULAR CLONING 72
7.1 Cleaving Of Dna Performed By Restriction Endonucleases Type Ii 73
7.2 Covalent Joining Of The Dna Fragments To The Vector Performed By Ligase 75
7.3 Cloning foreign DNA by adding linkers 76
7.4 Cloning foreign DNA by adding adaptors 77
7.5 Directional cloning: two different ERs 77
7.6 Plasmid Vectors 78
7.6.1 The ideal plasmid 79
7.6.2 First generation plasmids: pBR322 79
7.6.3 Second generation cloning vectors: pUC plasmids 79
7.6.4 Third generation cloning vectors: specialized plasmids 80
7.7 RECAP: Cloning a Piece of DNA 80

8. ENZYMES FOR MANIPULATING DNA 86

9. PCR: POLYMER CHAIN REACTION 91
9.1 DNA Replication In Vivo Vs PCR In Vitro 91
9.2 Limitations Of PCR 91
9.3 Advantage Of PCR 91
9.4 Working Principle Of PCR 92
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