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1 By: Mohsen Naeemipour In the name of God Molecular Biotechnology.

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1 1 By: Mohsen Naeemipour In the name of God Molecular Biotechnology

2 2 Timetable of Course The Development of Molecular Biotechnology DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals

3 3 Lecture 4: Chemical Synthesis, Amplification, and Sequencing of DNA

4 44 Chemical Synthesis of DNA

5 5 Assembling whole genes or parts of genes Amplifying specific DNA sequences Introducing mutations into cloned genes Screening gene libraries Sequencing DNA Facilitating gene cloning

6 6 Flowchart for the chemical synthesis of DNA oligonucleotides

7 7 Spacer molecule controlled-pore glass (CPG) bead, dimethoxytrityl (DMT) Starting complex for the chemical synthesis of a DNA strand

8 8 A Phosphoramidite Molecular

9 9 An anhydrous reagent (acetonitrile) Argon Trichloroacetic acid (TCA) (Detritylation) Acetonitrile Argon

10 10 Activation and coupling phosphoramidite tetrazole

11 11 Acetic anhydride and dimethylaminopyridine are added to acetylate the unreacted 5′ hydroxyl groups

12 12 The phosphite triester is oxidized with an iodine mixture to form the more stable pentavalent phosphate triester

13 13 Flowchart for the chemical synthesis of DNA oligonucleotides

14 14 Overall yields of chemically synthesized oligonucleotides with different coupling efficiencies

15 15 Uses of Synthesized Oligonucleotides

16 16 Typical linker and adaptor sequences

17 17 Cloning with a linker

18 18 Creating a restriction endonucleas e site in a vector with an adaptor

19 19 Enzymatic DNA synthesis of a gene

20 20 Assembly of a synthetic gene from short oligonucleotides

21 21 Assembly and in vitro enzymatic DNA synthesis of a gene

22 22 Gene Synthesis by PCR

23 23

24 24 DNA- Sequencing Techniques

25 25 Blocked DNA synthesis

26 26 A. dideoxynucleotide B. deoxyribonucleotide

27 27 Primer extension during DNA synthesis in the presence of dideoxynucleotides

28 28 Simulated autoradiograph of a dideoxynucleotide DNA- sequencing gel

29 29 Automated fluorescent- dye terminator Sanger DNA sequencing

30 30

31 31 DNA sequencing by primer walking

32 32 Timetable of Course The Development of Molecular Biotechnology DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals

33 33 Lecture 5: Manipulation of Gene Expression in Prokaryotes

34 34 Promoter and transcription terminator sequences Strength of the ribosome-binding site Number of copies of the cloned gene Gene is plasmid borne or integrated into the genome of the host cell Final cellular location of the synthesized foreign protein Efficiency of translation in the host organism Intrinsic stability within the host cell of the protein encoded by the cloned gene. Manipulation of Gene Expression

35 35 lac and trp (tryptophan) operons promoters are commonly used hybrid constructs the ratio of the number of repressor protein molecules to the number of copies of the promoter sequences two different plasmids repressor gene is placed on a low- copy-number (1-8) high-copy-number plasmid (30-100) Regulatable Promoters

36 36 Regulation of gene expression controlled by the pL promoter

37 37 A portion of the DNA sequence of the E. coli lac promoter (plac) and its mutated, more active, form (pmut).

38 38 pCP3 vector pPLc2833 plasmid + pKN402 Increasing Protein Production

39 39 Large-Scale Systems Dual-plasmid system for controlling the λ pL promoter by regulating the cI repressor with tryptophan

40 40 Uses of Fusion Proteins

41 41 Some protein fusion systems used to facilitate the purification of foreign proteins in E. coli and other host organisms

42 42 Schematic representation of the genetic construct used to produce a secreted fusion protein reducing the degradation enabling the product to be purified

43 43 Immunoaffinity chromatographic purification of a fusion protein

44 44 Purification of a protein

45 45 Surface Display

46 46 outer membrane protein A (OmpA). peptide- glycan-associated lipoprotein (PAL) from E. coli, Pseudomonas aeruginosa outer membrane protein F (OprF).

47 47 Translation Expression Vectors A ribosome-binding site is a sequence of 6 to 8 nucleotides (e.g., UAAGGAGG) in mRNA

48 48 Rarely codons used by the host cell (1)If the target gene is eukaryotic, it may be cloned and expressed in a eukaryotic host cell (2) A new version of the target gene containing codons that are more commonly used by the host cell may be chemically synthesized (codon optimization) (3) A host cell that has been engineered to overexpress several rare tRNAs may be employed

49 49 Increases in gene expression that result from altering the codon usage of the wild-type gene (or cDNA) to more closely correspond to the host E. coli cell

50 50 the Ara h2 protein, approximately 100- fold over the amount that was synthesized in conventional E. coli cells Overexpress several rare tRNAs AGG, AGA, AUA, CUA, and CGA

51 51 1. Intrinsic Protein Stability PEST sequences, are rich in proline (P), glutamic acid (E), serine (S), and threonine (T) Increasing Protein Stability Stability of B- galactosideas e with certain amino acids added to its N terminus

52 52 2. Facilitating Protein Folding Osmotic shock from E. coli cells into the growth medium. high temperatures (80°C) Enterokinase.

53 53 Disulfide bond formation in E. coli requires the participation of two soluble periplasmic enzymes (DsbA and DsbC) and two membrane bound enzymes (DsbB and DsbD).

54 54 Chaperonin 60 gene (cpn60) and the cochaperonin 10 gene (cpn10) Oleispira Antarctica (psychrophilic bacterium) Temperature-sensitive esterase (180- fold higher) 3. Coexpression Strategies

55 55 Use of Protease- Deficient Host Strains 4. Overcoming Oxygen Limitation One consequence of the stationary phase is the production by the host cells of proteases that can degrade foreign proteins. Use of Protease-Deficient Host Strains E. coli has at least 25 different proteases a housekeeping function RNA polymerase sigma factor secreted target proteins that had a 36- fold-greater specific activity Bacterial Hemoglobin (Vitreoscilla bacterium) a gram-negative obligate aerobe synthesize a hemoglobin-like molecule

56 56 Metabolic load A fraction of the cell population often loses its plasmids during cell growth. Cells that lack plasmids generally grow faster than those that retain them, Two methods of combating Growing the cells in the presence of either an antibiotic or an essential metabolite (industrial-scale) DNA Integration into the Host Chromosome

57 57 A generalized protocol for DNA integration includes the following steps. 1. Identify the desired chromosomal integration site, i.e., a segment of DNA on the host chromosome that can be disrupted without affecting the normal functions of the cell 2. Isolate and clone part or all of the chromosomal integration site 3. Ligate a cloned gene and a regulatable promoter either into or adjacent to the cloned chromosomal integration Site

58 58 A generalized protocol for DNA integration includes the following steps. 4. Transfer the chromosomal integration fragment–cloned-gene construct into the host cell as part of a plasmid that cannot replicate in the host cell 5. Select and perpetuate host cells that express the cloned gene Propagation of the cloned gene can occur only if it has been integrated into the chromosomal DNA of the host cell

59 59 DNA Integration into the Host Chromosome

60 60 Increasing Secretion Purification easier and less costly More stable Facilitates the correct formation of disulfide bonds (oxidative environment)

61 61 signal peptide (also called the signal sequence, or leader peptide) prlA4 and secE genes, which encode major components of the molecular apparatus that physically moves proteins across the membrane (50% to more than 90%) Secretion into the Periplasm

62 62 Yields of several secreted recombinant proteins produced in different bacteria

63 63 Schematic representation of protein secretion

64 64 Engineering the secretion of interleukin-2

65 65 Secretion into the Medium Host organisms gram-positive prokaryotes or eukaryotic cells, Genetic manipulation to engineer gram- negative bacteria Aspergillus Nidulans

66 66 Secretion into the Medium Bacteriocin release protein activates phospholipase A, which is present in the bacterial inner membrane, and cleaves membrane phosopholipids so that both the inner and outer membranes are permeabilized

67 67

68 68 YebF is naturally secreted to the medium without lysing the cells or permeabilizing the membranes

69 69 Metabolic load

70 70 Metabolic Load… An increasing plasmid copy number and/or size requires increasing amounts of cellular energy for plasmid replication and maintenance The limited amount of dissolved oxygen in the growth medium is often insufficient for both host cell metabolism and plasmid maintenance and expression.

71 71 Effect of plasmid copy number on host cell growth rate

72 72 Metabolic Load… Overproduction of both target and marker proteins may deplete the pools of certain aminoacyl-tRNAs (or even certain amino acids) and/or drain the host cell of its energy (in the form of ATP or GTP). When a foreign protein is overexpressed and then exported from the cytoplasm to the cell membrane, the periplasm, or the external medium, it may “jam” export sites and thereby prevent the proper localization of other, essential host cell proteins.

73 73 The foreign protein may interfere with the functioning of the host cell, for example, by converting an important and needed metabolic intermediate into a compound that is irrelevant, or even toxic, to the cell. Metabolic Load…

74 74 A decrease in the rate of cell growth particular aminoacyl- tRNA becomes limiting (1)The specific activity and stability of the target protein are significantly lowered (2) the incorrect amino acids may cause the protein to be immunogenic in humans. Metabolic Load…

75 75 Integrate the introduced foreign DNA directly into the chromosomal DNA of the host organism will not waste its resources synthesizing unwanted and unneeded antibiotic resistance marker gene products Metabolic Load

76 76 Timetable of Course The Development of Molecular Biotechnology DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals

77 77 Lecture 6: Heterologous Protein Production in Eukaryotic Cells

78 78 Desired biological activity or stability (posttranslational processing) Bacterial compounds that are toxic and pyrogens Disadvantages of prokaryote systems

79 79 disulfide bonds Disulfide Bonds Proteolytic incision Glycosylation, phosphorylation,… posttranslational modifications

80 80 Cleavage of inactive preproinsulin to yield active mature insulin

81 81 Examples of some O-linked oligosaccharide s in yeasts (A), insects (B), and mammals (C)

82 82 Generalized eukaryotic expression vector

83 83 Electroporation lithium acetate protoplast formation (treatment, and cell wall removal) Three techniques are commonly used to transform yeasts

84 84 The advantages of using Saccharomyces cerevisiae Biochemistry, genetics, and cell Biology Grown rapidly to high cell densities on relatively simple media in both small culture vessels and large-scale bioreactors Several strong promoters Many posttranslational modifications The product can be easily purified (yeast normally secretes so few proteins) Generally recognized as safe” organism

85 85 Recombinant proteins produced by S. cerevisiae expression systems

86 86 Episomal, or plasmid, vectors (yeast episomal plasmids [YEps]) Integrating vectors (yeast integrating plasmids [YIps]) YACs There are three main classes of S. cerevisiae expression vectors

87 87 S. cerevisiae expression vector

88 88 Schematic representation of integration of DNA with a YIp vector

89 89 Secretion of heterologous proteins by S. cerevisiae All glycosylated proteins of S. cerevisiae are secreted type α-factor gene (prepro-α- factor) Endoprotease that recognizes the dipeptide Lys-Arg The overproduction of molecular chaperones and protein disulfide isomerases

90 90 Summary of protein folding in the endoplasmic reticulum of yeast cells.

91 91 Pichia Pastoris Hansenula polymorpha Kluyveromyces Lactis Other Yeast Systems

92 92 P. pastoris integrating expression vector

93 93 Integration of DNA into a specific P. pastoris chromosome site

94 94 Baculovirus

95 95 Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)

96 96 Replacement of the AcMNPV polyhedrin gene

97 97 Production of recombinant baculovirus

98 98 Construction of a recombinant bacmid

99 99 Generalized mammalian expression vector

100 100 Two-vector expression system

101 Two-gene expression vector

102 102 Bicistronic expression vector

103 103 Timetable of Course The Development of Molecular Biotechnology DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals

104 104 Lecture 7: Directed Mutagenesis and Protein Engineering

105 105 Oligonucleotide-directed mutagenesis by M13

106 106 Enrichment of mutated M13 by passage of the parental DNA through a dut ung strain of E. coli dUTPase (dut). uracil N-glycosylase (ung).

107 107 Oligonucleotide-directed mutagenesis with plasmid DNA

108 108 Error-prone PCR of a target gene yields a variety of mutated forms of the gene

109 109 Random mutagenesis of a target DNA by using degenerate oligonucleotides and PCR

110 110 Timetable of Course The Development of Molecular Biotechnology DNA, RNA, and Protein Synthesis Recombinant DNA Technology Chemical Synthesis, Amplification, and Sequencing of DNA Manipulation of Gene Expression in Prokaryotes Heterologous Protein Production in Eukaryotic Cells Directed Mutagenesis and Protein Engineering Molecular Diagnostics Protein Therapeutics Nucleic Acids as Therapeutic Agents Bioinformatics, Genomics, and Proteomics Transgenic Animals

111 111 Lecture 8: Molecular Diagnostics

112 112 A comparison of some of the methods used to diagnose parasite infection

113 113 Generalized ELISA protocol for detecting a target antigen (Indirect Elisa) (Enzyme linked Immunosorbant assay)

114 114 Direct Elisa

115 115 Direct sandwich Elisa

116 116 Indirect sandwich Elisa

117 117 Schematic representation of a target antigen

118 118 The HAT procedure for selecting hybrid spleen–myeloma (hybridoma) cells hypoxanthine-guanine phosphoribosyltransferase (HGPRT−) hypoxanthine, aminopterin, and thymidine (HAT medium) dihydrofolate reductase

119 119 Screening for the production of a monoclonal antibody

120 120 Overview of the development and use of a DNA hybridization probe Diagnosis of Malaria (Plasmodium falciparum)

121 121 Chemiluminescent detection of target DNA

122 122 Southern blot of a forensic DNA sample

123 123 Detection of the sickle-cell anemia gene at the DNA level

124 124 PCR/OLA procedure (oligonucleotide ligation assay)


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