recombinat receptor expression systems in pharamcology

Question 1

What is a recombinant receptor?

Answer 1

A receptor (protein) deliberately generated under controlled experimental conditions

Question 2

What is recombinant protein?

Answer 2

DNA inserted into bacteria, theses bacteria will make protein based on this recombinant DNA. This protein is know as recombinant protein.

Question 3

What is over expression?

Answer 3

Vectors selected that will create huge quantities of preferred proteins

Question 4

What are the two methods for producing recombinant protein?

Answer 4

Molecular cloning

Polymerase chain reaction

Question 5

What is molecular cloning?

Answer 5

A laboratory method used to make recombinant DNA

Question 6

What is PCR?

Answer 6

A method to intensify a single or a few copies of a piece of DNA

Question 7

What is the difference between the two methods for producing recombinant protein?

Answer 7

Molecular cloning incorporates the replication of the DNA within a living cell, whereas PCR replicates DNA in the test tube, without living cells

Question 8

What is a vector?

Answer 8

A DNA molecule used as a vehicle to carry foreign genetic material into another cell where it can be replicated or expressed

Question 9

What are the applications of recombinant proteins?

Answer 9

Medical applications-medicines and vaccines

Human growth hormone, cell reproduction ect.

Question 10

What can recombinant DNA be used to recognize?

Answer 10

Map and sequence genes when scientists are learning separate genes or the entries genomes

Question 11

What are uses of recombinant proteins in regards to pharmacology experiments?

Answer 11

To investigate channel properties/receptor protein function

Question 12

What is transcription?

Answer 12

Transfer of information from DNA to mRNA

Question 13

What is translation?

Answer 13

the synthesis of protein based on sequence specified by mRNA

Question 14

What are advantages of prokaryotic receptor systems?

Answer 14

Fast
Inexpensive media
Easy to manipulate genetically
High yield

Question 15

Give examples of prokaryotic receptor systems

Answer 15

e-coli, baculovirus/insect cell

Question 16

Name a two eukaryotic receptor systems

Answer 16

Human embryonic kidney (HEK) 293 cells, yeast ect.

Question 17

what is the difference between prokaryotes and eukaryotes transcription/translation?

Answer 17

Prokaryotes- transcription and translation occur simultaneously
Eukaryotes- processes are separated with transcription in nucleus and translation in cytoplasm

Question 18

What is cell-based systems?

Answer 18

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.

Question 19

What is the advantage of bacterial expression?

Answer 19

Easily producing large amounts of protein, which required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination

Question 20

Why are eukaryotic proteins expressed in bacteria often non-functional?

Answer 20

Bacteria prokaryotes lack the full enzymatic machinery to accomplish post-translational modifications or molecular folding

Question 21

What are disadvantages of mammalian in vivo expression systems?

Answer 21

Low yield
Time-consuming
toxicity to host cells

Question 22

What are two prokaryotic systems?

Answer 22

Escherichia coli (e.coli)
Pseudomonas fluorescens

Question 23

What are pseudomonas fluorescens?

Answer 23

Non- pathogenic and gram-negative bacteria used for high of recombinant proteins. For vaccines

Question 24

What are two eukaryotic systems?

Answer 24

saccharomyces cerevisiae, Pichia pastors

baculovirus-infected cells

Question 25

What is non-lytic cell expression?

Answer 25

Vectors are transiently or stably transfected into the chromosomal DNA of insect cells for subsequent protein expression

Question 26

What is the advantage of non-lytic systems?

Answer 26

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

Question 27

What are pros of recombinant receptor?

Answer 27

produce large quantities of membrane receptor protein
Greater control of experimental conditions
Way to study specific activity

Question 28

What are cons of recombinant receptor?

Answer 28

Interference form endogenous receptors, time consuming process, unphysiological

Question 29

What does expression of recombinant receptors/proteins allow?

Answer 29

Investigate protein function, activation, regulation, inactivation, agonist potency , etc. in isolation

Question 30

What is cell culture?

Answer 30

Cells grown under controlled conditions generally outside of their natural environment

Question 31

Where do cells come from?

Answer 31

Purified from blood (red and white blood cells, platelets)
Derived from isolated tissue sections following enzymic digestion
explant culture

Question 32

What conditions are cells grown and maintained at?

Answer 32

37oC , 5% CO2 /95% O2 in a cell incubator

Question 33

What can variations of conditions for a particular cell type result in?

Answer 33

Different phenotypes

Question 34

What can vary in different cell growth medium?

Answer 34

pH
Glucose concentration 
Growth factors
Presence of other nutrients
Antibiotics

Question 35

What does aseptic technique avoid?

Answer 35

Contamination with bacteria, yeast or other cell lines

Question 36

Where are manipulations carried out?

Answer 36

In a biosafety hood or laminar flow

Question 37

Why are media changes carried out?

Answer 37

To remove media in which nutrients and growth hormones are depleted and toxic waste products have accumulated

Question 38

What is passaging cells?

Answer 38

Transfer of a small number of cells into a new vessel

Question 39

Why is passaging of cells done?

Answer 39

To avoid prolonged high cell density.

Without passaging mammalian cell lines will die due to a rise in toxic meatbolites

Question 40

List two issues of cells in culture dividing

Answer 40

cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop diving
cell-to-cell contact can stimulate cellular diffrnetiation

Question 41

What are the three manipulations of cell culture?

Answer 41

Media changes
Passaging cells
transfecting cells

Question 42

What is transfecting cells?

Answer 42

The process of deliberately introducing nucleic acids into cells used for non-viral methods in eukaryotic cells

Question 43

What does transfection of animal cells involve?

Answer 43

Opening transient pores or holes in the cell membrane to allow the uptake of genetic material

Question 44

What cam transfection be carried out using?

Answer 44

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

Question 45

What are the two types of transfection?

Answer 45

Transient transfection and generation of a stable cell line

Question 46

What is the difference between transient transfection and stable transfection?

Answer 46

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

Question 47

What is the Xenopus oocyte heterologous expression system?

Answer 47

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