Production of recombinant insulin requires an expression vector whcih includes promoter of the lac operon regulating beta-galactosidase gene fused to DNA sequence coding either A or B peptide of insulin. The fusion point between mentioned above DNA sequences has:
a. stop codon for beta-galactosidase gene
b. start codon for the insulin sequence part
c. to be in frame between beta-galactosidase gene and sequence coding for the insulin part.
D. a promoter for the insulin part
Real-time PCR is a powerful diagnostic tool. Using real time PCR one can measure the original amout of target DNA in the sample by
a. detecting how many cycles it takes to amplify specific amount of target DNA in comparison to standard DNA sample with known DNA concentration
b. measuring final yield of amplified target DNA in comparison to standard DNA sample with known DNA concentration
c. running electrophoresis and measuring the intesity of the DNA band containing target amlicon
To determine DNA sequence using Sanger, one needs to
a. perform 4 independant chemical reactions with the same DNA fragment in each case cleaving DNA at on of four nucleotides
b. design a DNA primer which will block DNA plymerase at one of four nucleotides
c. run four independent DNA polymerase reactions each containing one of four dideoxyribonucleotides.
d. fragment chromosomal DNA into pieces so that enormous chromosomal DNA size does not interfere with sequencing reaction
these are naturally occuring enzymes protecting host bacterial cells from foreign DNA and cut at palindromic sequences.
c-DNA libraries are used during new drug design projects. Creation of c-DNA library requires the action of this enzyme to convert mature mRNA sequences into DNA sequences
single nucleotide insertion in the protein coding region of the gene cause this type of mutation.
circular DNA molecule containing replication origin and often used as a vector in biotech drugs.
transcription, translation, transport of mRNA from the nucleus to the cytoplasm, and mRNA degradation rates all effect the rate of this biologic process
A set of thousands of DNA segments from a genome, each carried by a plasmid, phage, or other cloning vector.
A gene library containing clones that carry complementary DNA (cDNA) inserts. The library includes only the genes that were transcribed in the cells whose mRNA was isolated to make the cDNA.
1. Denature protein at 95 C
2. Uaw primers (DNA polymerase requires Primers not RNA).
3. 65 C the DNA begins to anneal
4. 72 C the TAC polymerase will begin to occur and extend the primers in each direction.
5. Repeat the cycle, now at 60 C newannealing will occur and you get 4 templates instead of 2.
6. Large amplification reaction
advantages of this cloning and analytical process are that it is very sensitive, relatively fast, and produces large amounts of DNA
disadvantages of this cloning and analytical process are that it is very sensitive and not as good with longer DNA molecules. Cannot amplify the whole chromosome or genome. There is a limit to how long the DNA can be to amplify.
laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.
Dideoxynucleotides halt dna polymerization at each base generating sequences of various lenths that encompass the entire orginial sequence. Terminated fragments are electrophoresed andthe original sequence can be deduced.
mutation that affects a single nucleotide, usually by substituting one nucleotide for another
Single nucleotide polymorphism abbreviation. Refers to nucleotide substitutions in a gene from different individuals of the same species.
Sanger sequencing and other sequencing methods are ultimate proof of these genetic changes.
The most common type of mutation, a base-pair substitution in which the new codon makes sense in that it still codes for an amino acid.
A mutation that changes a single nucleotide, but does not change the amino acid created.
A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.
therapy where patients cells are removed, grown in culture, incubated with viral vector to introduce genes and transplanted back into patient
The natural conditions in which organisms reside. Refers to biological processes that take place within a living organism or cell under normal conditions
a short single stranded nucleic acid polymer. These are most commonly used to intervate in gene transcription, translation DNA repair, recombination, gene construction and different diagnostic analyses.
Folded RNAs that code for a particular protein; also can start and stop production of the protein
These are nucleic acid ligand binders. Th RNA and DNA can fold like proteins into proteins into defined 3d structurs. They can bind nucleic acid, proteins, small organic compounds of interest.
Enzyme-linked immunosorbent Assay. Proteins bound to the plastic microtiter dish, and then the well is treated with specific antibody linked to an enzyme generating a colored product. This is used to quantify antigen
This poly ethanol structure is used to stabilize proteins in solution so that they properly fold.
these types of instabilities consist of oxidation, hydrolysis of amide side chain, and hydrolysis of the peptide bond.
In these instabilities there is no actual change in chemical bonds. They consist of denaturation, aggregation, precipitation, and adhesion to surfaces.
A test for the presence of a substance using the reaction of an antibody to its antigen, making use of the high selectivity of components of biological immune systems.
The movement of suspended particles through a fluid or gel under the action of an electromotive force applied to electrodes in contact with the suspension.
Type of analysis used to separate proteins based on mass. SDS binds to the proteins, giving them a negative charge cancelling the effect of charges from the individual amino acids. Lighter proteins travel faster than heavier ones.
Type of chromatography used to separate proteins based on charge. Stationary gel has an established pH gradient and the mobile phase proteins will travel to the point where the pH equals their isoelectric point.
size exclusion chromatography
Small beads of polymerized glucose, agarose, or acrylamide are manufactured with different sizes of poe depending on the degree of cross-linking of the polymer.. In this the larger particles come out first
ion exchange chromatography
proteins have charges due to amino acid side groups, bind to charged column matrix depending on their charge at a particular pH, anionic:negatively charged matrix(Heparin Sepharose), cationic:positively charged matrix(Q sepharose), elute bound proteins based on charge__then diplace by salt or pH change
reverse phase chromatography
Column has hydrophobic groups on the side and pulls out particles based on their hydrophobic properties. High ionic strength buffers are needed to get the solution out of this chromatography
Chromatography method which takes advantage of a proteins affinity to a specific ligand. The most powerful separation technique for proteins. Could be a specific ligand in the chromatography column or could be an antibody and should have a specificity and it is hard to get antigen off of antibodies.
destroys the compound, use high speed beam of electrons to ionize sample (eject an electron), a particle accelerator put charged particles in flight, a magnetic field deflect the accelerated cationic fragments and a detector records the number of particles of each mass that exit deflector area
Animals that contain genes transferred from other animals, usually from a different species
genetically engineered mice in which a specific gene has been inactivated, for an example by introducing a deletion in its DNA.
the bioreactor that involves encapsulated cellsin carriers that are in the medium and this allows for continual washing with new medium
This bioreactor is literally a semi-permeable membrane between cell culture and fresh medium. Allows for exchange of nutrients, oxygen, and waste, but does not allow for the cells to cross to the other side.
This type of growth medium consists of putting all the ingredients together, mix it up, and let it grow, without adding anything new to it other than oxygen. No more added nutrients or removal of excrements .
This type of growth medium consists of a medium similar to batch, but in this one we are going to continue to add new medium until you reach a point when the culture reaches its maximum.
This type of growth medium is the most preferable which is not easy to do using a stir reactor, but if you use a semipermiable membrane you continue to wash, continue to keep it growing. This is very useful when you culture is actually secreting the protein.
Hybrid cell lines that make monoclonal antibodies of defined specificity. They are formed by fusing a specific antibody-producing B lymphocyte with a myeloma cell that grows in tissue culture and does not make any immunoglobulin chains of its own.
fetal calf serum
This is the magical elixir. Mammalian cells do not culture well until you add this.
Contaminants native to the cells themselves (examples: viruses, glycosylation variants, n and c terminal variants, and endotoxins)
Contaminants that are manipulated end products. Denatured, aggregated, fragmented, or misfolded forms of the produced protein.
Contaminants from the media or added substances in purification (examples: media components, fetal calf serum, purification reagents, metals, column materials).
Can be done by inactivation (heat, UV, dehydration, solvents, detergens or antibodies) or removal (chromatography, filtration, or selective precipitation methods)...
Particulate removal, concentration, protein capture/initial purification, intermediate purification, final purification, and sterilization and formulation.
This stage of downstream processing starts with samll amounts of the protein in the laboratory setting trying to make a purification process
This stage of the downstream process involves applying the design stage to larger quantities of protein.
Separation technique used to separate particles according to mass, shape and density. Greater mass and density settle near the bottom while lighter compounds remain on top. This is meant to simulate a gravitational pull.
the fractionation method that often follows differential centrifugation. Adding salt to the crude extract causes some proteins to be less soluble and be released
Chromatography with a polar stationary phase. Protein of interest binds to polar stationary phase and then released by converting the polar phase to non-polar. hydrophobic interaction or reverse phase chromatography
Indicates the protein has undergone degradation during purification steps. This can be done by endopeptidase or exopeptidases. If done by exopeptidases then it will have this type of heterogeneity.
both the N and C terminal ends of a protein can undergo degradation by _____. These are located in the hepatocytes
the most abundant of these are the storage granules that hold the cell's reserve supplies of nutrients. some species of bacteria store carbon, some poly-beta-hydroxyalkanes (carbon in granules composed of lipids), polyphosphate (phosphate...essential component for ATP). These form if the protein is produced in bacterial host cells and may result in denaturation of the protein products.
these methods of preparation strive for this. They include aseptic technique, autoclaves for equipment, filtration of product, gloves, masks, and coats.
This type of contaminant is a bacterial endotoxins. Lipopolysaccharides. Highly charged and have hydrophobic regions. Removed using gel filtration or ion exchange chromatography methods.
at what stage is it difficult to remove viral or viral particles from a solution. This is why you remove them early in the purification process.
any more or less inert substance added to a prescription in order to confer a suitable consistency or form to the drug; a vehicle; inactive ingredients
these excipients are needed depending on how many hydrophylic side chains are exposed. Include salts, buffers, detergents or surfactants
albumin or other inert proteins, detergents or surfactants. Excipients that help prevent proteins binding to surfaces or other proteins
Include ascorbic acid, sodium formaldehyde sulfoxylate, GSH. THey prevent the oxidation of S atoms.
used for multi-entry or multi-dose vials. Phenylmurcuric nitrate, thimerosol, beta-hydroxybenzoic acid, phenols, and small alcohols
capable of killing bacteria. Antibiotics, antiseptics, and disinfectants can be bactericidal.
substance that assist in hydrating and stabilizing proteins in solution. Salts, mono and di-saccharides, polyalcohols such as PEG.
1st step in freeze drying process. Need to worry about excipients crystallizing at different temperatures and thus causing possible instabilities to the protein
This is the second step of the freeze drying process. Below freezing temperature but the pressure is lowered drastically. As you are pulling of water molecules, what happens to the surface left behind? The evaporation of the sweating as the water molecules enter the gaseous state and the protein will decrease in temperature. So to maintain this temperature we gotta add a tiny bit of heat while lowering the pressure.
Last step of freeze drying. Final water content is brought to 1.0%. Brown sample means impurity. Need to start over
Often times starch. So if you have a hydrophilic drug and want to deliver it transdermally you need to encapsulate it with a lipophilic sphere and get it to go through the skin and then break down once in circulation.
Can encapsulate the proteins in biodegradable microsphers.
This recombinant protein can be formulated with metal ions especially zinc to form stable suspensions
open loop delivery
drug can be delivered at a fixed rate over time. Pulse rate, response rate, general rate. there is no direct feedback. We are either preprogramming a certain rate or manually giving more when we feel we need more. No feedback from blood levels
biodegradable polymer implants involve impregnating a ____ with the drug and letting it dissolve in the skin.
closed loop systems
Needs direct feedback. These are insulin pumps taht would need a direct feedback loop to adjust the dose in the patient.
antibody acts as a homing divice and has a toxin or radionuclide attached to it to do damage to specific cells
colloidal particulate carrier systems
consist of liposomes, nanospheres and microspheres, and LDL molecules
These colloidal particulate carrier systems encapsulate the drug and are targeted towards the liver. This is because it is a normal cholesterol derivative
Fatty layer. These colloidal particulate carrier systems can have a lipophilic drug in its bilayer and a lipophobic drug inside the capsule.
the study of the movement of drugs and their metabolites through the body from absorption to excretion (basically, what the body does to the drug)
disproportional growht; larger animals require less energy/unit mass vs smaller animals b/c of surface area; when animals double in size, the SA is squared, but the weight is cubed, so the SA cannot support the weight; the proportion is not right; proportion changes when animals change size
proteins less than 60 kDa goe through the glomerulus. 30kDa undergo extensive filtration.
This PK/PD model is related to the distribution of the drug. Because of its size, physiochemical properties, ability to stick, etc. the distribution is going to be different.
this PK/PD model is related to Receptor effects. Not related to the protein in the vascular system rather it's the effect of the protein actually reaching the receptor.
cell lifespan models
apply to cells in the blood. Some protein drugs specifically cause an increase in levels of blood cells, so measuring the number and half-life of blood cells shows the drug is having its effect.
central tolerance is mediated in bone marrow by binding self-proteins which leads to deletion or inactivation of immature b cells that express IgM that reacts with self