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

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

4. Chemical Synthesis of DNA4

5. Chemical Synthesis of DNA
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. Flowchart for the chemical synthesis of DNA oligonucleotides

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

8. A Phosphoramidite Molecular

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

10. Activation and coupling
phosphoramidite tetrazole

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

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

13. Flowchart for the chemical synthesis of DNA oligonucleotides

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

15. Uses of Synthesized Oligonucleotides

16. Typical linker and adaptor sequences

17. Cloning with a linker

18. Creating a restriction endonuclease site in a vector with an adaptor

19. Enzymatic DNA synthesis of a gene19

20. Assembly of a synthetic gene from short oligonucleotides

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

22. Gene Synthesis by PCR

24. DNA-Sequencing Techniques24

25. Blocked DNA synthesis

26. A. dideoxynucleotide
B. deoxyribonucleotide

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

28. Simulated autoradiograph of a dideoxynucleotide DNA-sequencing gel

29. Automated fluorescent-dye terminator Sanger DNA sequencing

31. DNA sequencing by primer walking

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. Expression in Prokaryotes
Lecture 5:
Manipulation of Gene
Expression in Prokaryotes

34. Manipulation of Gene Expression
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.

35. Regulatable Promoters
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)

36. Regulation of gene expression controlled by the pL promoter

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

38. pPLc2833 plasmid + pKN402
pCP3 vector
Increasing Protein Production

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

40. Uses of Fusion Proteins

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

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

43. Immunoaffinity
chromatographic
purification of
a fusion protein

44. Purification of a protein

45. Surface Display

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

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

48. Rarely codons used by the host cell
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. 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. Overexpress several rare tRNAs
the Ara h2 protein, approximately 100-fold
over the amount that was synthesized in conventional E. coli cells
AGG, AGA, AUA, CUA, and CGA

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

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

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

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
Use of Protease-Deficient Host Strains

56. DNA Integration into the Host Chromosome
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. 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. 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. DNA Integration into the Host Chromosome

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

61. Secretion into the Periplasm
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%)

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

63. Schematic representation of protein secretion

64. Engineering the secretion of interleukin-2

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

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

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

69. Metabolic load

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. Effect of plasmid copy number on host cell growth rate

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. Metabolic Load…
• 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.

74. Metabolic Load…
A decrease in the rate of cell growth particular aminoacyl-tRNA becomes limiting
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.

75. Metabolic Load
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

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. Heterologous Protein Production
Lecture 6:
Heterologous Protein Production
in Eukaryotic Cells

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

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

80. Cleavage of inactive preproinsulin to yield active mature insulin

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

82. Generalized eukaryotic expression vector

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

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. Recombinant proteins produced by S. cerevisiae expression systems

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

87. S. cerevisiae expression vector

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

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

91. Other Yeast Systems
Pichia Pastoris
Hansenula polymorpha
Kluyveromyces Lactis

92. P. pastoris integrating expression vector

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

94. Baculovirus

95. Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)

96. Replacement of the AcMNPV polyhedrin gene

97. Production of recombinant baculovirus

98. Construction of a recombinant bacmid

99. Generalized mammalian expression vector

100. Two-vector expression system

101. Two-gene expression vector

102. Bicistronic expression vector

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. Directed Mutagenesis and Protein
Lecture 7:
Directed Mutagenesis and Protein
Engineering

105. Oligonucleotide-directed mutagenesis by M13

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. Oligonucleotide-directed mutagenesis with plasmid DNA

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

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

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. Molecular Diagnostics
Lecture 8:
Molecular Diagnostics

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

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

114. Direct Elisa

115. Direct sandwich Elisa

116. Indirect sandwich Elisa

117. Schematic representation of a target antigen

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

119. Screening for the production of a monoclonal antibody

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

121. Chemiluminescent detection of target DNA

122. Southern blot of a forensic DNA sample

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

124. PCR/OLA procedure
(oligonucleotide ligation assay)