Recombinant Protein Expression; ELISA

Question 1

enzyme-linked immunosorbent assay (ELISA)

Answer 1

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)

Question 2

Primary Antibody (ELISA)

Answer 2

recognizes the target protein or antigen

Question 3

Secondary Antibody (ELISA)

Answer 3

recognize the primary antibody and carries a detection system

Question 4

TA cloning

Answer 4

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

Question 5

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

Answer 5

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.

Question 6

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

Answer 6

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)

Question 7

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

Answer 7

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

Question 8

Internal Ribosomal Entry Sites (IRESs)

Answer 8

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

Question 9

Describe the primers used to make PCR easier

Answer 9

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

Question 10

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

Answer 10

The polymerase primes synthesis from the 3’ end

Question 11

How are shuttle vectors used concerning mammalian cell cultures?

Answer 11

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

Question 12

Overlap PCR

Answer 12

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

Question 13

Genetecin or G-418

Answer 13

Aminoglycoside antibiotic that kills animal cells by blocking protein synthesis

Question 14

How does OVERLAP PCR amplification occur?

Answer 14

-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

Question 15

DHFR gene / dihydrofolate reductase

Answer 15

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.

Question 16

methotrexate

Answer 16

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

Question 17

methionine sulfoximine

Answer 17

Toxic analog of methionine

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

Question 18

baculoviruses

Answer 18

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

Question 19

polyhedrons

Answer 19

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

Question 20

polyhedrin

Answer 20

Protein that comprises polyhedron structure of baculoviruses

Question 21

multiple nucelar polyhedrosis virus (MNPV)n

Answer 21

A particular baculovirus widely used as a cloning vector

Question 22

bacmids

Answer 22

Hybrid cloning vector made from a baculovirus and a plasmid

Question 23

Directed mutagenesis

Answer 23

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

Question 24

How are insect cells used for recombinant proteins?

Answer 24

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.

Question 25

Expression Vectors

Answer 25

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

Question 26

Translational Expression Vectors

Answer 26

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

Question 27

Why is yeast useful for producing recombinant proteins?

Answer 27

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

Question 28

What do translational expression vectors contain?

Answer 28

• a convenient selective marker
• a strong, regulated transcription promoter

Question 29

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

Answer 29

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

Question 30

What can be a cause for protein production to slow?

Answer 30

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

Question 31

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

Answer 31

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.

Question 32

What can solve the problem of slowed protein production?

Answer 32

• Adding more of the rare tRNA
• Changing the rare codon wobble position

Question 33

What happens when too much protein is manufactured too fast?

Answer 33

The surplus forms inclusion bodies

Question 34

What are inclusion bodies?

Answer 34

dense crystals of misfolded and nonfunctional proteins

Question 35

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

Answer 35

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

(EX. pET and pBAD for E. coli)

Question 36

Where do recombinant proteins usually go once produced?

Answer 36

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

Question 37

type I secretory system

Answer 37

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

Question 38

Molecular Chaperones 🍕🍕🍕🍕

Answer 38

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

Question 39

type II secretory system

Answer 39

Specialized export system that spans the outer membrane only

Question 40

general secretory system

Answer 40

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

Question 41

What is another way to deal with protein misfolding

Answer 41

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

Question 42

How is secretion directed across the inner cytoplasmic membrane?

Answer 42

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.

Question 43

What methods exist to help arrange for proper protein secretion?

Answer 43

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

Question 44

How can you alter the stability of proteins?

Answer 44

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)

Question 45

PEST sequences

Answer 45

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