Unit 3 - Biotechnology & Gene cloning Flashcards
What two types of molecules are there for options in drug synthesis?
Macromolecules
- producing therapeutic proteins
Small chemical entities (SCE) drugs
- organic chemistry synthesis
What is a protein?
Polymer made up of smaller units called amino acids which are linked together in a specific sequence by peptide bonds
What are the four levels of protein structure?
Primary
Secondary
Tertiary
Quaternary
What is the primary structure of a protein?
Sequence of amino acids in a chain
What is the secondary structure of a protein?
Local folding of the polypeptide chain into helices or sheets
What is the tertiary structure of a protein?
Three dimensional folding pattern of a protein due to side chain interactions
What is the quaternary structure of a protein?
Protein consisting of more than one amino acid chain
What are used to produce macromolecules?
Prokaryotes - bacteria Eukaryotes - yeast - cell culture - transgenic plants - tobacco - transgenic animals - goats - sheep - cows
What are the advantages of using prokaryotes to produce macromolecules?
Easy to construct Supports large scale production Can express toxic proteins No ethical issues Low cost
What are the advantages of using eukaryotes to produce macromolecules?
Can perform some forms of post-translational modification
- glucosylation
What is a gene?
A sequence of DNA which encodes a protein
How can a gene be inserted into a bacterial or eukaryotic cell so that it is expressed?
Genetic engineering
Give some examples of therapeutic proteins made using genetic engineering
Insulin
Interferon
Monoclonal antibody
What are chimeric proteins?
Proteins that do not exist in nature
How are chimeric proteins made?
Created by joining two genes to make a single gene which encodes a protein with functional properties derived from each of the original proteins
How is a therapeutic protein made?
Cloning Expression Fermentation (mass production) Cell lysis Soluble/insoluble protein Refolding Purification Characterisation Activity assays, interaction analysis
How is the target gene isolated and cloned?
1.Harvest a copy of the gene using restriction enzymes
- Amplify a copy using Polymerase Chain Reaction
- PCR
3.Synthetically produce a copy
What is PCR used for?
To make a copy of the gene sequence
It also allows you to incorporate restriction sites at either end of the sequence.
This means you can generate any restriction sites you want.
What is the mechanism of DNA ligase?
To form two covalent phosphodiester bonds between 3’ hydroxyl ends of one nucleotide with 5’ phosphate ends of another
- ATP is required for the ligase reaction
What different methods can be used to introduce a vector and gene into a host bacterium?
CaP/heat shock
Electroporation
What different methods can be used to introduce a vector and gene into a host yeast?
Electroporation
DMSO
What different methods can be used to introduce a vector and gene into a host mammalian cells?
CaP
Electroporation
Lipofectin
What is electroporation?
Electrical shock makes cell membrane permeable to DNA
How can clones containing the desired gene be selected for?
Loss of Antibiotic resistance
Nutrient utilisation-colour change
Screen for the physical presence of the gene
Screen for expression of the gene product
Give an example of how a change of colour confirms insertion of a gene
LacZ gene for B-galactosidase inserted into plasmid
X-gal turns blue upon digestion
- modified galactose molecule
Blue colonies produced
- have taken up LacZ gene for B-galactosidase
Destroy the ability of the gene to make the enzyme and therefore the colonies can’t produce the enzyme and are white.
How can the physical presence of a gene be screened for?
Cloned gene inserted into plasmid Plasmid cut with Sal 1 Plasmid cut with EcoR1 Plasmid cut with Sal 1 and EcoR1 Electrophoresis carried out to show bands
How can the expression of the gene product be screened for?
Detect expression of protein on basis of size
Specific antibodies which recognise the protein
- Western blot
Detect the presence of expression tags which have been added to the protein during cloning process
Detect the biological activity
- of the recombinant protein
- enzymatic activity
- of the expression tag
What are the four steps to producing a therapeutic protein?
Transformation
- double stranded recombinant plasmid DNA introduced into bacterial cell
Selection
- isolate bacteria containing target DNA
Propagation
- cell culture produces hundreds of millions of new bacteria
Transfer to large scale expression host
- many copies of purified plasmid isolated from lysed bacteria
What are the stages in the production phase of production a therapeutic protein?
Transfer to large scale expression host Fermentation - large scale production Harvesting - cell lysing - soluble/insoluble proteins - refolding Purification Characterisation Activity assays, interaction analysis
What are the three steps in the manufacturing process of therapeutic proteins?
Grow
Lyse
Purify
What factors need to be considered when developing a large scale production process?
Recombinant protein expression system - seed stock - provenance of starting material Production media Downstream processing Production facilities - dedicated production facilities - bio-security Costs
How can recombinant proteins be harvested from bacterial host?
Secreted proteins - easy to purify Soluble proteins trapped inside cell - release by cell lysis Proteins can form insoluble inclusion bodies - release by cell lysis - refold as soluble protein
What can be used to lyse bacterial cells?
Enzymes
Sonication
French press
What are inclusion bodies?
Over expression of recombinant proteins in E.coli can lead to the formation of insoluble structures
How can therapeutic proteins be recovered from inclusion bodies in bacteria?
Denature the protein
- 6M urea
How do restriction enzymes work?
Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences.
Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs. However, some produce blunt ends.
Describe the Polymerase chain reaction
Heat the DNA to separate into two strands.
Add a primer which will stick to their complimentary DNA when the sample has cooled
reheat the sample to a slightly lower heat than before and use DNA polymerase(taq polymerase) to add Complimentary DNA nucleotides to the strands.
