Pharmaceutical BiotechnologyPart 1

Supervised by: Prof. DR. Zaki Abdul-GhaniDone by: Hussein Talal Ashour

ID. 201117011

Introduction :Biotechnology in pharmaceutical sciences

• Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bio products. Biotechnology also utilizes these products for manufacturing purpose.

• Pharmaceutical biotechnology involves the use of living organisms such as microorganisms to create new pharmaceutical products or more safer and cost effective ones.

• Genetic engineering involves altering DNA molecules outside an organism, making the resultant DNA molecules functional in living cells.

• Recombinant DNA technology: A series of procedures used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed from segments from 2 or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, autonomously or after it has become integrated into a chromosome.

Introduction :Biotechnology in pharmaceutical sciences

• The 1st recombinant pharmaceutical product was insulin

• Recombinant DNA biotech. Allows the production of attenuated, vector and DNA vaccines

• Gene therapy was one of the great potentials in pharmaceutical biotech. Which consists of the insertion of genetic material into cells to prevent, control or cure disease

• It encompasses repairing or replacing defective genes and making tumors more susceptible to other kinds of treatment

Enabling techniques

1) Cutting and joining DNA molecules

2) Cloning vectors

3) Introduction of vectors into the host

4) Construction of genomic libraries

5) Screening of genomic libraries

6) Optimizing expression of recombinant genes

7) Amplifying DNA: The PCR

Cutting and joining DNA molecules

• DNA isolated from any type of cell can be fragmented using restriction endonucleases

• Endonucleases are enzymes which cut DNA at specific points known as ”restriction sites”

• There are 2 different types of DNA ends that can be generated using restriction enzymes; these are sticky ends and blunt ends.

• DNA fragments that have been generated by restriction enzymes can be joined together using DNA ligase.

• Only blunt ends generated by any restriction enzymes or sticky end generated by identical restriction enzymes will be joined by DNA ligase

Cutting and joining DNA molecules

Table of some restriction enzymes

Cloning vectors

• A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted.

• The main type of vectors used for gene cloning are plasmid cosmids and bacteriophages, which are used according to the size of DNA fragments that need to be cloned into them.

• Plasmids are used for cloning of small fragments of DNA

• Bacteriophages and cosmids are used for cloning of large fragments of DNA

Plasmids

• Plasmids are small extrachromosomal DNA molecules that replicate independently using their own origin of replication.

• Features that are generally found in plasmids and are used as cloning vectors:

1. Antibiotic resistance markers

2. Multiple cloning sites: Fragments of DNA that contain a number of different restriction sites, enabling the use of a choice of restriction enzymes for the cloning of DNA fragments.

3. Origin of replication: required for plasmid replication in a specific host.

Plasmids

Plasmids

An ideal plasmid should

1. Be Small in size

2. Be conjugation-defective, i.e. non-metabolizable

3. Have ready selectable phenotype on host cells

4. Contain large number of single restriction sites and high copy number (more than 10 copies per cell)

Cloning of of large fragments of DNA

• Its used in cloning genes responsible for the synthesis of an antibiotic

• Sometimes synthesis of the antibiotics require more than 10 different genes

Bacteriophage

• Most popular bacteriophage used is E.coli lambda bacteriophage which is made of tubular protein tail and a protein head packed with approx. 50kb of DNA.

• After injection of the viral DNA into E.coli it can multiply and enter a lytic cycle leading to the lysis of the host cell and subsequent release of large no. of phage particles.

• Alternatively, injection of the DNA can lead into a lysogenic cycle in which the phage DNA is integrated into the E.coli chromosome where its maintained until environmental conditions change and is then excised , entering a lytic cycle

Bacteriophage

• Phage genome integrates by an attachment site (att) with a partially homologous site on the E. coli chromosome, where it replicates as a chromosomal DNA seg ment.

• In this pathway no phage structural proteins are synthesized.

• The interactions of two proteins, the cl genes expressed protein (by phage genome) and cro gene expressed protein (by E.coli chromosomes) decide between the events of the lytic and lysogenic pathways.

Bacteriophage

Following are the advantages of phage cloning system over the plasmids:

• (i) DNA can be packed in vitro into phage particles and transduced into E. coli with high efficiency,

• (ii) foreign DNA up to 25 Kb in length can be inserted into phage vector, and

• (iii) screening and storage of recombinant DNA is easier

Replication of phage lambda in a Escherichia coli cell. A - lytic cycle; 1. rolling circle replication; 2. production of concatemers; 3. cleavage at cos site; 4. transcription and translation; 5. packaging. B - lysogenic cycle.

Introduction of vector into host cells

• Four main methods used to achieve this are transformation, electropoartion, conjugation and transduction.

• For transformation bacteria such as E.coli can uptake recombinant plasmid DNA by treating the cells with ice-cold CaCl2 until they reach a competent state in which they are ready to take up DNA.

• In Electropoartion, DNA is introduced into both bacteria and eukaryotic cells. This technique is based on the induction of free DNA uptake by bacterium after subjecting it to a high electric field.

Construction of genomic libraries

• A genomic library is a set of recombinant clones that contain the entire DNA present in an individual organism.

• An E.Coli genomic library, for example, contains all the E.Coli genes, so any

desired gene can be withdrawn from the library and studied. • Genomic libraries can be retained for many years, and propagated so that

copies can be sent from research group to research group. • Genomic libraries are prepared by purifying total cell DNA and then making a

partial restriction digest.

• Then such recombinant vectors transformed into suitable host cells and they are cultured in suitable selective medium for recombinant vectors. Selected clones are screened for specific genes and they are labeled and maintained as library.

Construction of genomic libraries

Eukaryotic cDNA Libraries

• A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism.

• The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.

• cDNA is produced from fully transcribed mRNA found in the nucleus.

• cDNA is created from a mature mRNA from a eukaryotic cell with the use of an enzyme known as reverse transcriptase

Construction of genomic libraries

Eukaryotic cDNA Libraries

Human genomic libraries can be constructed using restriction nucleases and ligase. A genomic library comprises a set of bacteria, each carrying a different small fragment of

human DNA. For simplicity, cloning of just a few representative fragments (colored) is shown. In reality, all the gray DNA fragments will also be cloned .

Construction of genomic libraries

SHOTGUN LIBRARY:

• Shotgun library generally prepared for sequencing genomes because sequencing methods are capable of sequencing smaller fragments.

• Initially genome is fragmented using nebulizer (shot gun).

• Fragmented DNA coupled with vectors in the presence of DNA ligase.

• Recombinant vectors then transformed into E.Coli and cultured in selectable medium.

• From the colonies grown in the culture, DNA isolated from the vector and sequenced one by one.

• After sequencing, each colony with its genetic nature tagged and maintained as genome library.

Bacterial cells Eukaryotic cell

1-genes are grouped into operons 1-genes are organize in single And hence transcribed together In transcriptional units interrupted A single molecule of mRNA by introns

2-whole process of transcription 2-Transcription in nucleus, and translation take place in the eukaryotic mRNA is firstly cytoplasm modified by the addition of a methylated guanine and then by the splicing of the introns. the mature mRNA is then exported into the cytoplasm where its translated into proteins

Construction of genomic libraries

Which library is to be used

Cosmids and lambda bacteriophage libraries are used when:1. Larger insert size of DNA2. Lower no. of clones required to have full presentation of an entire genome3. Lower no. of recombinant clones to be screened

Plasmid libraries are used when:4. Smaller insert size5. Single gene is to be isolated

Cloning method suitabilty

Host Insert size (KB)

Vector

Shotgun cloningcDNA cloning

Specified by the origin of replication present on the plasmid

<10 plasmid

Shotgun cloning E.coli 7-22 Lambda bacteriophage

Shotgun cloning E.cloi 25-45 cosmids

Screening of genomic libraries

1-Hybridization screening• Used when DNA sequence of the gene is known • Used when fragment of such gene is available from previous cloning

2- Immunological screening• Used when we need to isolate a gene coding for protein for which anti

bodies available• The success of this technique relies on the expression of the gene of interest

3-protein activity screening• Its limited to proteins that have specific activity that can easily be identified• To detect protein activity, the gene coding for this protein must be

expressed and active protein must have been produced

Optimizing expression of recombinant genes

• The 1ry objective of pharmaceutical companies involved in the production of recombinant drugs is the maximal expression of recombinant genes to generate large quantities of these drugs

• To improve the expression of a gene, we have to optimize the different stages that lead to the synthesis of proteins

• This can be achieved by

1. Optimizing transcription: where the recombinant gene must be preceded by promoter and followed by transcriptional terminator

2. Optimizing translation: the nucleotide sequence of the ribosome binding site (RBS), located upstream of the gene needs to be efficiently recognized by the ribosomes of the host, in addition the distance between the RBS and the translation start codon needs to be optimal to lead to the synthesis of protein

3. Posttranslational modifications: such as correct s-s bond formation, photolytic cleavage of precursor, glycosylation, phosphorylation ….. etc

Amplifying DNA: the polymerase chain reaction (PCR)

• This technique allows the generation of large amounts of copies of a specified DNA sequence from single DNA molecule without the need for cloning.

• The PCR exploits certain characteristic of DNA replication, as it uses single-stranded DNA as a template for the synthesis of complementary new stands.

• The single-stranded DNA templates can be generated by heating the double-stranded DNA to 90 Celsius.

• DNA polymerase requires small fragments of double-stranded DNA to initiate DNA synthesis .

• The PCR reaction uses special DNA polymerase that can withstand temperatures as high as 99 Celsius, working optimally at 72 Celsius and subsequently reducing the risk of mismatches that occasionally occur at lower temperatures.

Advantages and disadvantages of PCR

Advantages1. Specificity2. Rapid technique (few hours)3. Versatility4. Inexpensive

Disadvatages5. Require knowledge of DNA sequence6. Amplify only large fragments (up to 20 kb)7. Contamination may give false positive results

Thank you

References1-Hugo and Russell's Pharmaceutical Microbiology 7th edition

2-http://en.wikipedia.org/wiki/Polymerase_chain_reaction

3-http://learnsomescience.com/wp-content/uploads/2011/05/PCR-cycle.gif

4-http://biosiva.50webs.org/dna%20cl9.jpg

5-http://en.wikipedia.org/wiki/CDNA_library