Kareem_Pharma_Biotech_Topic_3.pptx PDF

Course contents Pharmaceuticals, biologics and biopharmaceuticals CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Protein structure Gene manipulation and recombinant DNA technology The drug development process Sources of biopharmaceuticals & upstream processing  Procaryotic Ce...

Course contents Pharmaceuticals, biologics and biopharmaceuticals CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Protein structure Gene manipulation and recombinant DNA technology The drug development process Sources of biopharmaceuticals & upstream processing  Procaryotic Cells, mammalian Cells ,Plants, insects, and transgenic Animals BRINGING THE DRUG INTO ACTION Downstream processing Product analysis RECENT APPLICATIONS IN PHARMACEUTICAL BIOTECHNOLOGY Metabolic Engineering of Medicinal Plants Metabolomics as Bioanalytical Tool Nutraceuticals/Functional Foods for Improving Health and Preventing Disease CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Gene manipulation and recombinant DNA technology  The biopharmaceutical sector is largely based upon the application of techniques of molecular biology and genetic engineering for the manipulation and production of therapeutic macromolecules.  The majority of approved biopharmaceuticals are proteins produced in engineered cell lines by recombinant means. Examples include the production of insulin in recombinant E. coli and recombinant S. cerevisiae, as well as the production of EPO (Erythropoietin) in an engineered animal cell line.  Molecular biology: describes the study of biology at a molecular level, but focuses in particular upon the structure, function and interaction/relationship between DNA, RNA and proteins.  Genetic engineering: describes the process of manipulating genes (outside of a cell’s/organism’s normal reproductive process). It generally involves the isolation, manipulation and subsequent reintroduction of DNA into cells and is usually undertaken in order to confer on the recipient cell the ability to produce a specific protein, such as a biopharmaceutical.  rDNA technology: is a term used interchangeably with ‘genetic engineering’. rDNA is a piece of DNA artificially created in vitro which contains DNA (natural or synthetic) obtained from two or more sources.  When developing a new protein biopharmaceutical, one of the earliest actions undertaken involves: 1. Identifying and isolating the gene or complementary DNA (cDNA) coding for the target protein. 2. Generation of an appropriate piece of rDNA containing the protein’s coding sequence 3. Introduction of this rDNA into an appropriate host cell such that the target protein is made in large quantities by that engineered cell. Recombinant production of therapeutic proteins  Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much. Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as DNA cloning. Brief Introduction Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology  The basic concepts for recombinant DNA technology  In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born— recombinant DNA technology.  Concept of Recombinant DNA Recombinant DNA is a molecule that combines DNA from two sources. The process is known as gene cloning. Creates a new combination of genetic material – Human gene for insulin was placed in bacteria – The bacteria are recombinant organisms and produce insulin in large quantities for diabetics – Genetically engineered drug in 1986 Genetically modified organisms are possible because of the universal nature of the genetic code!  Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment( how? ), regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became possible.  Recombinant technology begins with the isolation of a gene of interest (target gene). The target gene is then inserted into the plasmid or phage (vector) to form replicon. The replicon is then introduced into host cells to be cloned and either express the protein or not. The cloned replicon is referred to as recombinant DNA. The procedure is called recombinant DNA technology. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells. Some other terms are also in common use to describe genetic engineering. Gene manipulation Recombinant DNA technology Gene cloning (Molecular cloning) Genetic modification  Cloning In classical biology, a clone is a population of identical organisms derived from a single parental organism. For example, the members of a colony of bacterial cells that arise from a single cell on a petri plate are clones. Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original molecule or cell. How recombinant technology works Steps include isolating of the target gene and the vector, specific cutting of DNA at defined sites, joining or splicing of DNA fragments, transforming of replicon to host cell, cloning, selecting of the positive cells containing recombinant DNA, and either express or not in the end. Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage, digestion) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology  The basic procedures of recombinant DNA technology  Six basic steps are common to most recombinant DNA experiments 1. Isolation and purification of DNA. Both vector and target DNA molecules can be prepared by a variety of routine methods. In some cases, the target DNA is synthesized in vitro. 2. Cleavage of DNA at particular sequences. As we will see, cleaving DNA to generate fragments of defined length, or with specific endpoints, is crucial to recombinant DNA technology. The DNA fragment of interest is called insert DNA. In the laboratory, DNA is usually cleaved by treating it with commercially produced nucleases and restriction endonucleases. 3. Ligation of DNA fragments. A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are typically joined using DNA ligase (also commercially produced). – T4 DNA Ligase 4. Introduction of recombinant DNA into compatible host cells. In order to be propagated, the recombinant DNA molecule (insert DNA joined to vector DNA) must be introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host cell is called genetic transformation (or transformation). Recombinant DNA can also be packaged into virus particles and transferred to host cells by transduction. 5. Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts. 6. Identification of host cells that contain recombinant DNA of interest. Vectors usually contain easily scored genetic markers, or genes, that allow the selection of host cells that have taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional step— screening a large number of recombinant DNA clones. This is almost always the most difficult step. DNA cloning in a plasmid vector permits amplification of a DNA fragment.  Isolating DNA 1. Vector 2. Target gene  How to get a target genes? Genomic DNA Artificial synthesis PCR amplification RT-PCR  Polymerase chain reaction (PCR) A technique called the polymerase chain reaction (PCR) has revolutionized recombinant DNA technology. It can amplify DNA from as little material as a single cell and from very old tissue such as that isolated from Egyptian mummies, a frozen mammoth, and insects trapped in ancient amber.  method is used to amplify DNA sequences  The polymerase chain reaction (PCR) can quickly clone a small Initial DNA segment sample of DNA in a test tube Number of DNA molecules PCR primers  RT-PCR Reverse transcription polymerase chain reaction (RT- PCR) is a variant of polymerase chain reaction (PCR. In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional PCR. – Template: RNA – Products: cDNA  Vectors- Cloning Vehicles Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes. Most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell, whereas a minority carry sequences that facilitate integration into the host genome.  All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA. The most useful vectors have several restriction sites grouped together in a multiple cloning site (MCS) called a polylinker.  Types of vector 1. Plasmid Vectors 2. Bacteriophage Vectors 3. Virus vectors 4. Shuttle Vectors--can replicate in either prokaryotic or eukaryotic cells. 5. Yeast Artificial Chromosomes as Vectors  Plasmid Vectors Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell. Plasmid  Plasmid vectors can replicate autonomously within a host, and they frequently carry genes conferring resistance to antibiotics such as tetracycline, ampicillin, or kanamycin. The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not  pBR322 pBR322 was one of the first versatile plasmid vectors developed; it is the ancestor of many of the common plasmid vectors used in biochemistry laboratories. pBR322 contains an origin of replication (ori) and a gene (rop) that helps regulate the number of copies of plasmid DNA in the cell. There are two marker genes: confers resistance to ampicillin, and confers resistance to tetracycline. pBR322 contains a number of unique restriction sites that are useful for constructing recombinant DNA.  pBR322 1. Origin of replication 2. Selectable marker 3. unique restriction sites  Enzymes 1. Restriction endonuclease, RE 2. DNA ligase 3. Reverse transcriptase 4. DNA polymerase, DNA pol 5. Nuclease 6. Terminal transferase Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into Cloning Vectors Restriction enzymes cleave DNA The same sequence of bases is found on both DNA strands, but in opposite orders. GAATTC CTTAAG This arrangement is called a palindrome. Palindromes are words or sentences that read the same forward and backward. form sticky ends: single stranded ends that have a tendency to join with each other ( the key to recombinant DNA) Restriction Enzymes Cut DNA Chains at Specific Locations Restriction enzymes are endonucleases produced by bacteria that typically recognize specific 4 to 8bp sequences, called restriction sites, and then cleave both DNA strands at this site. Restriction sites commonly are short palindromic sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 5′ → 3′ direction. Cut out the gene Restriction enzymes  Restriction enzymes Restriction enzymes are named after the bacterium from which they are isolated – For example, Eco RI is from Escherichia coli, and Bam HI is from Bacillus amyloliquefaciens. The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discovery (eg, EcoRI, EcoRII).  Blunt ends or sticky ends Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4–8 bp long. These DNA cuts result in blunt ends (eg, Hpa I) or overlapping (sticky) ends (eg, BamH I) , depending on the mechanism used by the enzyme. Sticky ends are particularly useful in constructing hybrid or chimeric DNA molecules. Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive ends (A) or blunt ends (B). This is an important consideration in devising cloning strategies. Inserting DNA Fragments into Vectors DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligases. For purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. The vector DNA and restriction fragment are covalently ligated together through the standard 3 → 5 phosphodiester bonds of DNA. DNA ligase “pastes” the DNA fragments together Ligation of restriction fragments with complementary sticky ends. Identification of Host Cells Containing Recombinant DNA Once a cloning vector and insert DNA have been joined in vitro, the recombinant DNA molecule can be introduced into a host cell, most often a bacterial cell such as E. coli. In general, transformation is not a very efficient way of getting DNA into a cell because only a very small percentage of cells take up recombinant DNA. Consequently, those cells that have been successfully transformed must be distinguished from the vast majority of untransformed cells.  Identification of host cells containing recombinant DNA requires genetic selection or screening or both. In a selection, cells are grown under conditions in which only transformed cells can survive; all the other cells die. In contrast, in a screen, transformed cells have to be individually tested for the presence of the desired recombinant DNA. Normally, a number of colonies of cells are first selected and then screened for colonies carrying the desired insert.  Selection Strategies Use Marker Genes (Primary screening) Many selection strategies involve selectable marker genes— genes whose presence can easily be detected or demonstrated. ampR Selection or screening can also be achieved using insertional inactivation.  insertional inactivation Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert.  Screening (Strategies) 1. Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments 2. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method 3. The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture 4. Blotting Techniques Permit Detection of Specific DNA Fragments and mRNAs with DNA Probes A B C M bp —1534 — 994 — 695 — 515 — 377 — 237 Gel Electrophoresis negative charged DNA run to the anode Sequencing results Southern blot technique can detect a specific DNA fragment in a complex mixture of restriction fragments. Hybridization Radioactive isotope  Types of blotting techniques Southern blotting Southern blotting techniques is the first nucleic acid blotting procedure developed in 1975 by Southern. Southern blotting is the techniques for the specific identification of DNA molecules. Northern blotting Northern blotting is the techniques for the specific identification of RNA molecules. Western blotting Western blotting involves the identification of proteins. Antigen + antibody  Expression of Proteins Using Recombinant DNA Technology Cloned or amplified DNA can be purified and sequenced, used to produce RNA and protein, or introduced into organisms with the goal of changing their phenotype. One of the reasons recombinant DNA technology has had such a large impact on biochemistry is that it has overcome many of the difficulties inherent in purifying low-abundance proteins and determining their amino acid sequences.  Recombinant DNA technology allows the protein to be purified without further characterization. Purification begins with overproduction of the protein in a cell containing an expression vector. – Prokaryotic Expression Vectors – Eukaryotic Expression Vectors  Prokaryotic Expression Vectors Expression vectors for bacterial hosts are generally plasmids that have been engineered to contain appropriate regulatory sequences for transcription and translation such as strong promoters, ribosome-binding sites, and transcription terminators.  Eukaryotic proteins can be made in bacteria by inserting a cDNA fragment into an expression vector. Large amounts of a desired protein can be purified from the transformed cells. In some cases, the proteins can be used to treat patients with genetic disorders. For example, human growth hormone, insulin, and several blood coagulation factors have been produced using recombinant DNA technology and expression vectors.  Expression of Proteins in Eukaryotes Prokaryotic cells may be unable to produce functional proteins from eukaryotic genes even when all the signals necessary for gene expression are present because many eukaryotic proteins must be post- translationally modified.  Several expression vectors that function in eukaryotes have been developed. These vectors contain eukaryotic origins of replication, marker genes for selection in eukaryotes, transcription and translation control regions, and additional features required for efficient translation of eukaryotic mRNA, such as polyadenylation signals and capping sites. pcDNA™4/HisMax Is designed for overproduction of recombinant proteins in mammalian cell lines