EXAM 3 - Session 24: Recombinant DNA Technology and Molecular Engineering Flashcards
Explain the 3 steps of recombinant DNA technology.
- Gene isolation
- Cloning and expression
- Protein production
Describe gene isolation.
Start with DNA then…
1) DNA –> mRNA –> protein –> aa sequencing –> DNA synthesis –> cDNA (desired gene)
2) DNA –> mRNA –> reverse transcriptase –> cDNA (desired gene)
3) DNA –> genomic library –> DNA probe –> cDNA (desired gene)
Because of the human genome project –> you can just order cDNA of the gene of interest
Explain cloning and expression.
Used to make multiple copies of the foreign DNA fragment (gene of interest) and then expressed to produce the gene product of interest.
* gene region of interest can be inserted into plasmid –> then inserted into bacteria cell –> bacteria cell replicates and replicates the foreign material too
Describe characteristics of plasmids (vectors).
- origin of replication
- promoter - makes sure the DNA is expressed in the cell
- restriction sites - where we put the foreign DNA
- antibiotic resistance - marker
Explain the factors that contribute to choosing the right vector/plasmid.
- endonucleases compatibility
- antibiotic resistance
- solubility
- stability
- purification
- expression in MM for NMR
Describe restriction endonucleases/enzymes.
A protein produced by bacteria that cleaves DNA at specific sites.
* Each restriction enzyme recognizes a short, specific sequence of nucleotide bases
* palindromic - reads the same forward and backwards
Describe the process of polymerase chain reaction PCR.
To increase amount of DNA
1. denature DNA double-helix at 90-94C
2. anneal (recombine) forward and reverse primers to target gene
3. extend primers by DNA polymerase in 5’-3’ direction on each strand
4. process repeats for exponential amplification
Explain the process of adding foreign DNA to plasmid vectors.
Foreign DNA is cut at each end with the same restriction enzyme.
* There will be sticky ends after cuts –> sticky ends are complementary and can bind together to form a circle (noncovalently)
* transforms into bacteria cell –> antibiotic resistance selection
* protein production (translation)
Explain the steps of bacterial protein production.
Inoculum
* start with cell bank and inoculate fermentor
cell culture
* create crude bulk cell paste
purification
* purify bulk protein
formulation
* form ready-to-use vials
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Explain the steps of mammalian protein production.
Inoculum
* start with cell bank and inoculate roller bottles
cell culture
* create crude bulk protein
purification
* purify bulk protein
formulation
* form ready-to-use vials
Describe the methods of making biologicals on-Demand.
- pichia pastoris yeast - as the host system to produce the protein of interest
- BioMod
List the methods of molecular engineering of proteins.
- amino acid sequence modification
- truncation
- glycosylation
- pegylation
- fusion molecule
- humanization
List the purposes of molecular engineering of proteins.
Describe glycoproteins.
- Membrane bound
- secreted proteins with attached oligosaccharides
- altered polarity and solubility
- influenced folding
- influenced tertiary structure
- protection from proteolytic enzymes
- specific interaction
Explain methods of purification.
- affinity based chromotagraphy
- cleavage fo the tag
- ion exchange
- size-exclusion
- reverse-phase
Describe glycosylation.
Wide variety of functions involving cell-cell recognition.
* the sialic acid cap determines the lifetime of the proteins in the bloodstream
Describe asailoglycoprotein.
Receptor removes immunoglobulins and peptide hormones that display terminal galactose residues rather than sialic acid residues.
Describe pegylation.
Ex. Prgfilgrastim - modified human granulocyte coloni-stimulating factor (G-CSF is used to treat neutropenias and infections)
- polyethylene glycol molecule added to improve stability of protein
- polyethylene glycol molecule added
- greatly increase stability
- dosing - 1 dose/cycle of chemotherapy vs. daily
- small fluctuations in plasma concentrations
- slowed clearance of protein
- clearance primarily by neutrophils
- possible enhanced activity
- possible decreased toxicity
Describe Naloxegol.
- many drugs modified with PEGylation
intranasal spray of pegylated opioid antagonist
- small molecule (all other biologicals)
- pegylation prevents drug from crossing the blood-brain barrier (polar)
- functions as a peripherally-acting mu-opioid receptor antagonist in tissues such as GI decreasing constipation effects of opioid
Describe the primary targets for HIV infection.
Primary targets of HIV infection:
* mononuclear phagocyte system
* t lymphocytes
* b lymphocytes
* natural killer lymphocytes
* dendritic cells
* hematopoietic stem cells
* endothelial cells.
* microglial cells
* gastrointestinal epithelial cells
List some sites of drug targeting for HIV.
- Entry/fusion inhibitors
- reverse transcriptase inhibitors
- integrase inhibitors
- protease inhibitors
Explain the significance of the Gp41-gp120 interface.
HIV-1 neutralization by a human antibody that binds interface
- prevents the folding back
- broad and extremely potent HIV-specific monoclonal antibody binds a novel HIV-1 envelope glycoprotein epitope
- trimmers gp41-gp120 interface and neutralizes HIV virus ability to enter the cell
Explain the drug action of Selzentry.
Inhibition of viral fusion
- binds to the core-ceptor CCR5 on T cells, thereby blocking HIV’s gp120 protein from associating with that coreceptor
- protein-protein interaction
- small molecule drug
