Recombinant Protein Expression; ELISA Flashcards
enzyme-linked immunosorbent assay (ELISA)
Sensitive method to assay the amount of a specific protein in a sample using enzyme-linked antibodies aka determines concentration of the antigen (target protein)
Primary Antibody (ELISA)
recognizes the target protein or antigen
Secondary Antibody (ELISA)
recognize the primary antibody and carries a detection system
TA cloning
Using Taq polymerase to generate single 3′-A overhangs on the ends of DNA segments that are used to clone DNA into a vector with matching 3′-T overhangs
What is the issue with mammalian proteins that have multiple subunits that have to assemble within a mammalian cell?
Each subunit must be made separately, but you cannot manufacture the separate subunits in separate cultures and then mix them together. It does not yield an active protein.
What are the three ways that more than on polypeptide can be synthesized in the same cell?
1) two separate vectors are used
2) one vector is use carrying two separate genes (each has its own promoter)
3) a single vector with an artificial operon (two genes with the same promoter)
How do primers make PCR easier when cloning a foreign piece of DNA?
PCR primers generate new restriction enzyme sites at the ends of the target sequence
Internal Ribosomal Entry Sites (IRESs)
Sequence allowing the translation of multiple coding sequences on the same message in a eukaryotic cell. IRES sequences are found on some animal viruses
Describe the primers used to make PCR easier
5’ end - has the desired restriction enzyme site
3’ end - has a sequence complementary to the target
Why is Taq Polymerase not bothered by mismatched 5’ sequences?
The polymerase primes synthesis from the 3’ end
How are shuttle vectors used concerning mammalian cell cultures?
Mammalian shuttle vectors have features for producing recombinant proteins in cultures mammalian cells as they can replicate in mammalian and bacterial cells.
Overlap PCR
PCR technique that uses overlapping primers to match small regions of two different gene segments
(used in joining segments of DNA from different sources)
Genetecin or G-418
Aminoglycoside antibiotic that kills animal cells by blocking protein synthesis
How does OVERLAP PCR amplification occur?
-One primer complementary to beginning of 1st gene segment
-Second primer complementary to end of 2nd gene
-Third primer half complementary to end of 1st gene segment and half complementary to beginning of 2nd gene segment
DHFR gene / dihydrofolate reductase
Enzyme that takes part in one carbon metabolism and is needed for the synthesis of thymine and adenine and protects mammalian cells from methotrexate.
Mammalian cells lacking the DHFR gene are used for selection and a functional copy of the DHFR gene is provided on the shuttle vector.
methotrexate
Antibiotic that inhibits the enzyme dihydrofolate reductase in animal cells
manipulation of methotrexate levels allows for corresponding increase or decrease in copy number off the vector
methionine sulfoximine
Toxic analog of methionine
Vectors use the glutamine synthetase gene as protection from methionine sulfoximine.
baculoviruses
Family of DNA viruses that infect insects and related invertebrates and are widely used as vectors