Recombinant Protein Expression; ELISA Flashcards

1
Q

enzyme-linked immunosorbent assay (ELISA)

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Primary Antibody (ELISA)

A

recognizes the target protein or antigen

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Secondary Antibody (ELISA)

A

recognize the primary antibody and carries a detection system

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

TA cloning

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the issue with mammalian proteins that have multiple subunits that have to assemble within a mammalian cell?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the three ways that more than on polypeptide can be synthesized in the same cell?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How do primers make PCR easier when cloning a foreign piece of DNA?

A

PCR primers generate new restriction enzyme sites at the ends of the target sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Internal Ribosomal Entry Sites (IRESs)

A

Sequence allowing the translation of multiple coding sequences on the same message in a eukaryotic cell. IRES sequences are found on some animal viruses

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe the primers used to make PCR easier

A

5’ end - has the desired restriction enzyme site
3’ end - has a sequence complementary to the target

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Why is Taq Polymerase not bothered by mismatched 5’ sequences?

A

The polymerase primes synthesis from the 3’ end

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How are shuttle vectors used concerning mammalian cell cultures?

A

Mammalian shuttle vectors have features for producing recombinant proteins in cultures mammalian cells as they can replicate in mammalian and bacterial cells.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Overlap PCR

A

PCR technique that uses overlapping primers to match small regions of two different gene segments
(used in joining segments of DNA from different sources)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Genetecin or G-418

A

Aminoglycoside antibiotic that kills animal cells by blocking protein synthesis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does OVERLAP PCR amplification occur?

A

-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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

DHFR gene / dihydrofolate reductase

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

methotrexate

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

methionine sulfoximine

A

Toxic analog of methionine

Vectors use the glutamine synthetase gene as protection from methionine sulfoximine.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

baculoviruses

A

Family of DNA viruses that infect insects and related invertebrates and are widely used as vectors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

polyhedrons

A

In reference to viruses, the packages of virus particles embedded in a protein matrix that are formed by baculoviruses

20
Q

polyhedrin

A

Protein that comprises polyhedron structure of baculoviruses

21
Q

multiple nucelar polyhedrosis virus (MNPV)n

A

A particular baculovirus widely used as a cloning vector

22
Q

bacmids

A

Hybrid cloning vector made from a baculovirus and a plasmid

23
Q

Directed mutagenesis

A

Deliberate alteration of the DNA sequence of a gene
(Used in PCR to change nucleotides and increase stability of annealed primers)

24
Q

How are insect cells used for recombinant proteins?

A

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.

25
Q

Expression Vectors

A

Vector designed to enhance gene expression (provides a strong promoter that drives the expression of the cloned gene)

26
Q

Translational Expression Vectors

A

Vector designed to enhance gene expression at the level of translation
(maximizes the initiation of translation)

27
Q

Why is yeast useful for producing recombinant proteins?

A

1) considered true eukaryotes
2) have plasmid vectors
3) grow fast and easily
4) have well-known characteristics

28
Q

What do translational expression vectors contain?

A
  • a convenient selective marker
  • a strong, regulated transcription promoter
29
Q

What are the three main classes of vectors that are used with yeast?

A

1) Plasmid Vectors
2) Vectors that integrate into the yeast chromosomes
3) Yeast artificial chromosomes

30
Q

What can be a cause for protein production to slow?

A

When the sequence for the recombinant protein encodes a rarely used tRNA

31
Q

What are the issues associated with producing recombinant proteins with yeast and their low yield?

A

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.

32
Q

What can solve the problem of slowed protein production?

A
  • Adding more of the rare tRNA
  • Changing the rare codon wobble position
33
Q

What happens when too much protein is manufactured too fast?

A

The surplus forms inclusion bodies

34
Q

What are inclusion bodies?

A

dense crystals of misfolded and nonfunctional proteins

35
Q

How do recombinant proteins control when and how much protein the host cell makes?

A

Expression systems - switch expression on/off and control amount made

(EX. pET and pBAD for E. coli)

36
Q

Where do recombinant proteins usually go once produced?

A

Recombinant proteins are usually exported out of the cell into the culture medium. The use of bacterial secretory systems facilitates the export.

37
Q

type I secretory system

A

Specialized export system that spans both inner and outer membranes of gram-negative bacteria such as E. coli

38
Q

Molecular Chaperones 🍕🍕🍕🍕

A

Proteins that oversees the correct folding of other proteins
(attach to polypeptides while being translated and keep protein from being folded)

39
Q

type II secretory system

A

Specialized export system that spans the outer membrane only

40
Q

general secretory system

A

Standard system for exporting proteins across membranes that is found in most organisms

41
Q

What is another way to deal with protein misfolding

A

Accept the formation of inclusion bodies and attempt refolding of the protein

42
Q

How is secretion directed across the inner cytoplasmic membrane?

A

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.

43
Q

What methods exist to help arrange for proper protein secretion?

A

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

44
Q

How can you alter the stability of proteins?

A
  1. Alter the identity of the N-terminal amino acid to change the half-life
  2. Remove certain internal sequences that destabilize the protein (PEST sequences)
45
Q

PEST sequences

A

regions of 10-60 amino acids that are rich in P (proline), E (glutamate), S (serine), and T (threonine)