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
polyhedrons
In reference to viruses, the packages of virus particles embedded in a protein matrix that are formed by baculoviruses
polyhedrin
Protein that comprises polyhedron structure of baculoviruses
multiple nucelar polyhedrosis virus (MNPV)n
A particular baculovirus widely used as a cloning vector
bacmids
Hybrid cloning vector made from a baculovirus and a plasmid
Directed mutagenesis
Deliberate alteration of the DNA sequence of a gene
(Used in PCR to change nucleotides and increase stability of annealed primers)
How are insect cells used for recombinant proteins?
They are easy to grow and can be infected with a virus genome that has the recombinant protein gene. Instead of making viral particles, the infected insect cell will make recombinant protein.
Expression Vectors
Vector designed to enhance gene expression (provides a strong promoter that drives the expression of the cloned gene)
Translational Expression Vectors
Vector designed to enhance gene expression at the level of translation
(maximizes the initiation of translation)
Why is yeast useful for producing recombinant proteins?
1) considered true eukaryotes
2) have plasmid vectors
3) grow fast and easily
4) have well-known characteristics
What do translational expression vectors contain?
- a convenient selective marker
- a strong, regulated transcription promoter
What are the three main classes of vectors that are used with yeast?
1) Plasmid Vectors
2) Vectors that integrate into the yeast chromosomes
3) Yeast artificial chromosomes
What can be a cause for protein production to slow?
When the sequence for the recombinant protein encodes a rarely used tRNA
What are the issues associated with producing recombinant proteins with yeast and their low yield?
1) expression plasmids are lost
2) proteins meant for secretion do not exit the space between the membrane and wall
3) glycosylation of proteins is excessive and the product will have too many sugar residues attached to be functional.
What can solve the problem of slowed protein production?
- Adding more of the rare tRNA
- Changing the rare codon wobble position
What happens when too much protein is manufactured too fast?
The surplus forms inclusion bodies
What are inclusion bodies?
dense crystals of misfolded and nonfunctional proteins
How do recombinant proteins control when and how much protein the host cell makes?
Expression systems - switch expression on/off and control amount made
(EX. pET and pBAD for E. coli)
Where do recombinant proteins usually go once produced?
Recombinant proteins are usually exported out of the cell into the culture medium. The use of bacterial secretory systems facilitates the export.
type I secretory system
Specialized export system that spans both inner and outer membranes of gram-negative bacteria such as E. coli
Molecular Chaperones 🍕🍕🍕🍕
Proteins that oversees the correct folding of other proteins
(attach to polypeptides while being translated and keep protein from being folded)
type II secretory system
Specialized export system that spans the outer membrane only
general secretory system
Standard system for exporting proteins across membranes that is found in most organisms
What is another way to deal with protein misfolding
Accept the formation of inclusion bodies and attempt refolding of the protein
How is secretion directed across the inner cytoplasmic membrane?
by a hydrophobic signal sequence at the N-terminal end of the newly synthesized protein. signal sequence is cut off after export by signal peptidase.
What methods exist to help arrange for proper protein secretion?
1) a signal sequence is engineering into the cloned gene
2) the recombinant protein may be fused to a bacterial protein that is normally exported
3) the gene of interest can be expressed in gram-positive bacteria
4) secretion across both membranes of gram-negative bacteria
How can you alter the stability of proteins?
- Alter the identity of the N-terminal amino acid to change the half-life
- Remove certain internal sequences that destabilize the protein (PEST sequences)
PEST sequences
regions of 10-60 amino acids that are rich in P (proline), E (glutamate), S (serine), and T (threonine)
