By: Mohsen Naeemipour

transcript

By:Mohsen Naeemipour

In the name of God

Molecular Biotechnology

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

Lecture 4:

Chemical Synthesis, Amplification, and Sequencing of

Chemical Synthesis of

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

Flowchart for the chemical synthesis of DNA oligonucleotides

Spacer moleculecontrolled-pore glass (CPG) bead, dimethoxytrityl (DMT)

Starting complex for thechemical synthesis of a DNA strand

A Phosphoramidite Molecular

An anhydrous reagent (acetonitrile)ArgonTrichloroacetic acid (TCA) (Detritylation)AcetonitrileArgon

Activation and coupling

phosphoramidite tetrazole

Acetic anhydride and dimethylaminopyridine are addedto acetylate the unreacted 5′ hydroxyl groups

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

Flowchart for the chemical synthesis of DNA oligonucleotides

Overall yields of chemically synthesized oligonucleotides withdifferent coupling efficiencies

Uses of Synthesized Oligonucleotides

Typical linker and adaptor sequences

Cloning with a linker

Creating a restriction endonuclease site in a vector with an adaptor

Enzymatic DNA synthesis of a

Assembly of a synthetic gene from short oligonucleotides

Assembly and in vitro enzymatic DNA synthesis of a gene

Gene Synthesis by PCR

DNA-Sequencing Techniques

Blocked DNA synthesis

A. dideoxynucleotideB. deoxyribonucleotide

Primer extension during DNA synthesis in the presence of dideoxynucleotides

Simulated autoradiograph of a dideoxynucleotide DNA-sequencing gel

Automated fluorescent-dye terminator Sanger DNA sequencing

DNA sequencing by primer walking

Timetable of Course

Lecture 5:

Manipulation of GeneExpression in Prokaryotes

• 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

• 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

Regulation of gene expression controlled by the pL promoter

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

pCP3 vector

pPLc2833 plasmid + pKN402

Increasing Protein Production

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

Uses of Fusion Proteins

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

Schematic representation of the genetic construct used to produce asecreted fusion protein

• reducing the degradation• enabling the product to

be purified

Immunoaffinity chromatographic purification of a fusion protein

Purification of a protein

Surface Display

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

Translation Expression Vectors

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

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 codonsthat 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

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

the Ara h2 protein, approximately 100-foldover the amount that was synthesized in conventional E. coli cells

Overexpress several rare tRNAs

AGG, AGA, AUA, CUA, and CGA

1. Intrinsic Protein StabilityPEST sequences, are rich in proline (P), glutamicacid (E), serine (S), and threonine (T)

Increasing Protein Stability

Stability of B-galactosidease with certainamino acids added to its N terminus

2. Facilitating Protein Folding

Osmotic shock from E. coli cells into the growth medium.high temperatures (80°C) Enterokinase.

Disulfide bond formation in E. coli requires the participationof two soluble periplasmic enzymes (DsbA and DsbC) and two membrane boundenzymes (DsbB and DsbD).

• Chaperonin 60 gene (cpn60) and the cochaperonin 10 gene (cpn10) Oleispira Antarctica (psychrophilic bacterium)

• Temperature-sensitive esterase (180-fold higher)

3. Coexpression Strategies

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 functionRNA 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

• Metabolic load• A fraction of the cell population often loses

its plasmids during cell growth.• Cells that lack plasmids generally grow

faster thanthose that retain them,

• Two methods of combatingGrowing the cells in the presence of either an antibiotic or an essential metabolite (industrial-scale)DNA Integration into the Host Chromosome

DNA Integration into the Host Chromosome

A generalized protocol forDNA 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

A generalized protocol forDNA 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 genePropagation of the cloned gene can occur only if it has been integrated into the chromosomal DNA of the host cell

DNA Integration into the Host Chromosome

Increasing Secretion

Purification easier and less costly

More stable

Facilitates the correct formation of disulfide bonds (oxidative environment)

• 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

Yields of several secreted recombinant proteins produced in different bacteria

Schematic representation of protein secretion

Engineering the secretion of interleukin-2

Secretion into the Medium

• Host organisms gram-positive prokaryotes or

eukaryotic cells,

• Genetic manipulation to engineer gram-negative bacteria

• Aspergillus Nidulans

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

YebF is naturally secreted to the medium withoutlysing the cells or permeabilizing the membranes

Metabolic load

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.

Effect of plasmid copy number on host cell growth rate

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.

• 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…

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…

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

Timetable of Course

Lecture 6:

Heterologous Protein Production in Eukaryotic Cells

• Desired biological activity or stability (posttranslational processing)

• Bacterial compounds that are toxic and pyrogens

Disadvantages of prokaryote systems

• disulfide• bonds

• Disulfide Bonds

• Proteolytic incision

• Glycosylation, phosphorylation,…

posttranslational modifications

Cleavage of inactive preproinsulin to yield active mature insulin

Examples of some O-linkedoligosaccharides in yeasts (A), insects (B), and mammals (C)

Generalized eukaryotic expression vector

• Electroporation

• lithium acetate

• protoplast formation (treatment, and cell wall removal)

Three techniques are commonly used to transform yeasts

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

Recombinant proteins produced by S. cerevisiae expression systems

• 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

S. cerevisiae expression vector

Schematic representation of integration of DNA with a YIp vector

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

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

• Pichia Pastoris

• Hansenula polymorpha

• Kluyveromyces Lactis

Other Yeast Systems

P. pastoris integrating expression vector

Integration of DNA into a specific P. pastoris chromosome site

Baculovirus

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)

Replacement of the AcMNPV polyhedrin gene

Production of recombinant baculovirus

Construction of a recombinant bacmid

Generalized mammalian expression vector

Two-vector expression system

Two-gene expression vector

Bicistronic expression vector

Timetable of Course

Lecture 7:

Directed Mutagenesis and Protein Engineering

Oligonucleotide-directed mutagenesis by M13

Enrichment of mutated M13 by passage of the parental DNA through adut ung strain of E. coli

dUTPase (dut).uracil N-glycosylase (ung).

Oligonucleotide-directed mutagenesis with plasmid DNA

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

Random mutagenesis of a target DNA by using degenerate oligonucleotidesand PCR

Timetable of Course

Lecture 8:

Molecular Diagnostics

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

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

Direct Elisa

Direct sandwich Elisa

Indirect sandwich Elisa

Schematic representation of a target antigen

The HAT procedure for selecting hybrid spleen–myeloma (hybridoma) cellshypoxanthine-guanine

phosphoribosyltransferase(HGPRT−)hypoxanthine, aminopterin, and thymidine (HAT medium)dihydrofolate reductase

Screening for the production of a monoclonal antibody

Overview of the development and use of a DNA hybridization probe

Diagnosis of Malaria(Plasmodium falciparum)

Chemiluminescent detection of target DNA

Southern blot of a forensic DNA sample

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

PCR/OLA procedure(oligonucleotide ligation assay)

By: Mohsen Naeemipour

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