recombinat receptor expression systems in pharamcology Flashcards
What is a recombinant receptor?
A receptor (protein) deliberately generated under controlled experimental conditions
What is recombinant protein?
DNA inserted into bacteria, theses bacteria will make protein based on this recombinant DNA. This protein is know as recombinant protein.
What is over expression?
Vectors selected that will create huge quantities of preferred proteins
What are the two methods for producing recombinant protein?
Molecular cloning
Polymerase chain reaction
What is molecular cloning?
A laboratory method used to make recombinant DNA
What is PCR?
A method to intensify a single or a few copies of a piece of DNA
What is the difference between the two methods for producing recombinant protein?
Molecular cloning incorporates the replication of the DNA within a living cell, whereas PCR replicates DNA in the test tube, without living cells
What is a vector?
A DNA molecule used as a vehicle to carry foreign genetic material into another cell where it can be replicated or expressed
What are the applications of recombinant proteins?
Medical applications-medicines and vaccines
Human growth hormone, cell reproduction ect.
What can recombinant DNA be used to recognize?
Map and sequence genes when scientists are learning separate genes or the entries genomes
What are uses of recombinant proteins in regards to pharmacology experiments?
To investigate channel properties/receptor protein function
What is transcription?
Transfer of information from DNA to mRNA
What is translation?
the synthesis of protein based on sequence specified by mRNA
What are advantages of prokaryotic receptor systems?
Fast
Inexpensive media
Easy to manipulate genetically
High yield
Give examples of prokaryotic receptor systems
e-coli, baculovirus/insect cell
Name a two eukaryotic receptor systems
Human embryonic kidney (HEK) 293 cells, yeast ect.
what is the difference between prokaryotes and eukaryotes transcription/translation?
Prokaryotes- transcription and translation occur simultaneously
Eukaryotes- processes are separated with transcription in nucleus and translation in cytoplasm
What is cell-based systems?
contain a combination of: an expression vector, its cloned DNA (cDNA), and the host for the vector to allow foreign gene function in a host cell and protein production. Over expression occurs.
What is the advantage of bacterial expression?
Easily producing large amounts of protein, which required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination
Why are eukaryotic proteins expressed in bacteria often non-functional?
Bacteria prokaryotes lack the full enzymatic machinery to accomplish post-translational modifications or molecular folding
What are disadvantages of mammalian in vivo expression systems?
Low yield
Time-consuming
toxicity to host cells
What are two prokaryotic systems?
Escherichia coli (e.coli) Pseudomonas fluorescens
What are pseudomonas fluorescens?
Non- pathogenic and gram-negative bacteria used for high of recombinant proteins. For vaccines
What are two eukaryotic systems?
saccharomyces cerevisiae, Pichia pastors
baculovirus-infected cells
What is non-lytic cell expression?
Vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent protein expression
What is the advantage of non-lytic systems?
Hither protein yeid
Quicker expression of recombinant proteins compared to baculovirus-infeted cell expression.
Cells do not lye and so several cultivation modes can be used
What are pros of recombinant receptor?
produce large quantities of membrane receptor protein
Greater control of experimental conditions
Way to study specific activity
What are cons of recombinant receptor?
Interference form endogenous receptors, time consuming process, unphysiological
What does expression of recombinant receptors/proteins allow?
Investigate protein function, activation, regulation, inactivation, agonist potency , etc. in isolation
What is cell culture?
Cells grown under controlled conditions generally outside of their natural environment
Where do cells come from?
Purified from blood (red and white blood cells, platelets)
Derived from isolated tissue sections following enzymic digestion
explant culture
What conditions are cells grown and maintained at?
37oC , 5% CO2 /95% O2 in a cell incubator
What can variations of conditions for a particular cell type result in?
Different phenotypes
What can vary in different cell growth medium?
pH Glucose concentration Growth factors Presence of other nutrients Antibiotics
What does aseptic technique avoid?
Contamination with bacteria, yeast or other cell lines
Where are manipulations carried out?
In a biosafety hood or laminar flow
Why are media changes carried out?
To remove media in which nutrients and growth hormones are depleted and toxic waste products have accumulated
What is passaging cells?
Transfer of a small number of cells into a new vessel
Why is passaging of cells done?
To avoid prolonged high cell density.
Without passaging mammalian cell lines will die due to a rise in toxic meatbolites
List two issues of cells in culture dividing
cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop diving
cell-to-cell contact can stimulate cellular diffrnetiation
What are the three manipulations of cell culture?
Media changes
Passaging cells
transfecting cells
What is transfecting cells?
The process of deliberately introducing nucleic acids into cells used for non-viral methods in eukaryotic cells
What does transfection of animal cells involve?
Opening transient pores or holes in the cell membrane to allow the uptake of genetic material
What cam transfection be carried out using?
Calcium phosphate, by electroporation, by cell squeezing or by mixing a actinic lipid with the material to produce liposomes which fuse with the cell membrane and deposit their cargo inside
What are the two types of transfection?
Transient transfection and generation of a stable cell line
What is the difference between transient transfection and stable transfection?
Transient expression means cell line is transformed in a way that the gene is only expressed for a short period of time whereas stable expression means the genes has been incorporated into a chromosome so that the transformed cell and all of its descendants will express the gene
What is the Xenopus oocyte heterologous expression system?
the oocyte is a major expression system used in membrane protein structure-function studies. Especially for expression of membrane transport proteins such as carriers and ion channels
