434 Exam 2

What is recombinant DNA?

Any DNA formed by joining DNA segments from different sources

What are the advantages to using recombinant DNA?

• Source availability - low natural abundance

• Safety - transmission of disease or dangerous sources

• Engineering - engineer for PK and PD

• Economics - cheaper to manufacture biologics

What is DNA digestion?

When restriction endonucleases cut DNA at specific internal sequences

What are restriction endonucleases?

Enzymes that cleave DNA at specific internal sequences

What are sticky DNA ends?

Ends of DNA with an overhang due to digestion from restriction endonucleases

How can 2 pieces of DNA be joined together using a restriction enzyme and DNA ligase?

If they have the same restriction site on each end , they can be digested with the same restriction enzyme and joined with DNA ligase

When reading a gel, will large DNA fragments be at the top or the bottom of the gel and why?

The top; large DNA fragments move slower through the gel

What is annealing?

The formation of a DNA double helix from two single stranded DNAs

What factors affect the annealing process

Strand length, pH, salinity, temperature

What is polymerase chain reaction (PCR)?

The process of amplifying genes based on base pairing specificity

What are the steps to PCR?

• Heat to denature

• Add primers and cool to anneal

• Add DNA polymerase to extend

How many cycles are typically run in PCR?

30 cycles

What is DNA ligase?

An enzyme that permanently joins cut DNA fragments

Do agarose gels perform electrophoresis with large or small DNA fragments?

Large fragments (>500 bp)

Do polyacrylamide gels perform electrophoresis with large or small DNA fragments?

Small fragments (<500 bp)

What is cDNA complementary to?

mRNA

What is reverse transcription?

The process that generates a cDNA from an mRNA

What is a structural gene?

A gene that codes for nonregulatory proteins that have activity in metabolism or other processes

What is a regulatory gene?

A gene that codes for a protein that binds DNA and influences transcription of structural genes

What is an operator sequence?

The sequence of DNA that a regulatory protein binds to

What is a promoter sequence?

The DNA sequence that RNA polymerase binds to in absence of a repressor

What protein does the lacZ gene code for?

Beta-galactosidase

What is the function of beta-galactosidase?

Degrade lactose

What function does the lacZ gene serve?

Encodes for a protein that degrades lactose

What protein does the lacY gene code for?

Galactoside permease

What is the function of galactoside permease?

Transporting lactose across the cell membrane

What purpose does the lacY gene serve?

Codes for a membrane protein to intake lactose into the cell

What protein does lacl code for?

The lac repressor

What is the function of the lac repressor?

Binds to the operator sequence upstream of the structural genes and blocks transcription

Is lacl a structural gene or a regulatory gene?

Regulatory gene

Is lacZ a structural gene or a regulatory gene?

Structural gene

Is lac Y a structural gene or a regulatory gene?

Structural gene

What is a plasmid?

A piece of circular DNA that replicates in bacteria and can be used for recombinant DNA cloning

What are ribosome binding sites (RBS)?

Sequences where the ribosome binds mRNA before translation

What is the function of erythropoietin (EPO)?

Stimulate the production of red blood cells (erythrocytes) from stem cell precursors

What is the purpose of recombinant human antithrombin (ATryn)?

Anti-clotting agent

What animal is recombinant human antithrombin (ATryn) transgenically produced from?

Goats

What is a transgenic organism?

A subset of GMOs which have inserted DNA that originated in a different species

What is the purpose of a helper plasmid?

Produce accessory proteins required for AAV packaging

What is an adeno associated virus (AAV)?

Small viruses with ssDNA genomes (~4.7 kb) that can insert their DNA specifically at one site in human chromosomal DNA

What is a polylinker (multi-cloning site)

A plasmid sequence with many restriction endonuclease sites for cloning recombinant DNAs; Allows the selective cutting of DNA based on which restriction enzymes are expressed

What is a pyrogen?

A bacterial toxin that can induce fever

What is an inclusion body?

An aggregate of misfolded protein in bacteria

What is glycosylation?

A post translational modification that adds complex sugars (glycans) to proteins

What are Chinese hamster ovary cells used to produce?

EPO and other glycosylated proteins

What are human embryonic kidney (HEK293) cells used to produce?

EPO and other glycosylated proteins

What is the function of an EPO receptor?

Bind to EPO and signal downstream pathways involved in

erythrocyte precursor differentiation

What is the purpose of the origin of replication?

Maintenance of plasmid DNA in bacteria; dictates the number of plasmid copies per cell

Why are bacterial vectors given antibiotic resistance genes?

To select for bacteria retaining the desired plasmid

What is the T7 promoter recognized by?

T7 RNA polymerase

What is the purpose of the Ni2+ column?

Purify proteins that are translationally fused to a His-tag

What is electroporation?

A transformation method that uses an electric current to insert DNA into expression hosts

What does mature insulin lack?

The N-terminal signal sequence and C-peptide chain

What is proinsulin?

An insulin precursor that lacks the N-terminal signal sequence

What is preproinsulin?

An insulin precursor that contains an N-terminal signal sequence, C-peptide, and the A-chain and B-chain

What are the A/B-chains?

Peptide components that have activity as mature insulin

What is insulin lispro (Humalog)?

The first approved mutein, rapid acting insulin, faster onset of action due to less aggregation at the injection site

What was the first approved mutein?

Insulin lispro (Humalog)

Why does insulin lispro have a faster onset of action?

Less aggregation at the injection site

What is a mutein?

A protein with an altered amino acid sequence; enough to alter its physicochemical and biological activity

What is the function of the N-terminal signal sequence?

Direct a protein to the endoplasmic reticulum

What is the PolyA tract?

A feature of mammalian expression plasmids that increases mRNA stability for the cloned gene

What is a lentivirus?

A subset of retroviruses that has a genome made of ssRNA (HIV)

What is the function of the Dicer protein?

Process double stranded siRNA precursors

What do insulin receptors signal?

Downstream pathways involved in cell growth

What part of the proinsulin protein is cleaved out before forming the mature hormone?

The C-peptide chain

What is a translation suppression oligo (TSO)?

An antisense oligo that binds tightly to mRNA adjacent to translational start sites and physically blocks ribosomal protein synthesis

How is recombinant DNA cloned?

• Plasmid is cut with 2 different restriction enzymes to remove a section

• Desired gene and plasmid are amplified

• Primers added to plasmid

• Desired gene is digested to have sticky ends complementary to the plasmid

• Gene is joined to plasmid using DNA ligase

• Plasmid is inserted into E. coli in the presence of an anitbiotic

What induces the expression of lacZ or B-galactosidase?

The presence of lactose

How is the lac operon used to express foreign DNA?

Genes of interest can be inserted after the promoter and expressed as a structural gene

What is the purpose of IPTG?

Block the expression of the repressor to enable expression of the structural genes

Why is a polylinker useful?

Gene of interest may contain a restriction enzyme site, resulting in it being cut if that restriction enzyme is used; Offers choices of restriction enzymes to use in case one doesn’t work

Why is the expression of T7 RNA polymerase regulated?

Too much is toxic for the cell

How is the expression of T7 RNA polymerase controlled?

Using the lac expression system.

• Genomic DNA: In the presence of IPTG, Ec RNA polymerase transcribes the T7 RNA polymerase gene.

• Plasmid DNA: T7 RNA polymerase binds to the T7 promoter and transcribes the therapeutic gene.

Why is T7 promoter used instead of the lac promoter?

T7 RNA polymerase produces more mRNA than the E. coli RNA polymerase

What is a pET vector?

A plasmid containing a T7 promoter upstream of the cloned gene of interest

Why would E. coli turn off expression of insulin?

Insulin is not useful for the cell

What is Cas9?

A CRISPR-associated protein that can be delivered to target genes by sgRNAs where it introduces a double-strand break in DNA

What is a single guide RNA (sgRNA)?

A fusion of natural CRISPR RNAs that simplifies genome editing

What kind of RNA is used to deliver Cas9 to target genes?

sgRNA

What is E. coli BL21(DE3)?

An E. coli expression host that contains T7 RNA polymerase to expresses genes cloned into a pET vector

Why is the expression of insulin so tightly controlled?

To prevent Ec from developing mutations that would inactivate the expression of inslulin

How do nickel columns purify DNA?

Nickle has an affinity for histidine residues (his tags) which are bound to the target protein; The desired protein is added to the column and gets stuck while the other junk is washed away

What is used to remove his-tagged proteins removed from the Ni2+ column?

Imidizole

How is the his-tag removed from a protein?

Protease

What are the advantages of prokaryotic bacteria?

• fast and easy to grow

• use of multiple expression vectors

• tags allow for purification and better protein solubility, folding, and secretion

What are the disadvantages of prokaryotic bacteria

• no post-translational modifications

• surface antigens

• accumulation of inclusion bodies from misfolding

What are the advantages of eukaryotic yeast?

• established genetics

• good protein production

• cheap and fast growth

• post-translational modification

What are the disadvantages of eukaryotic yeast?

• hyperglycosylation (mannose)

• low protein yield

What are the advantages of mammalian cells?

• post-translational modification (glycosylation)

What are the disadvantages of mammalian cells?

• low concentrations of protein

• retrovirus contamination

Where does N-glycosylation take place?

Endoplasmic reticulum

How does glycosylation contribute to correct folding?

Glucose acts as a folding signal

Where does modification of proteins take place?

ER and golgi apparatus

What is the purpose of glycosylation?

Ensure correct protein folding and dictate where the protein is sent

What is site-directed mutagenesis?

The process used to make single mutations in cloned genes and generate muteins

Why is human antithrombin (ATryn) produced in mammalian cells?

It is heavily glycosylated

What holds the A and B chains together?

Disulfide bonds

What happens when insulin binds to its receptor?

• Causes conformation change in the receptor

• Beta-chain tyrosine kinase is activated by self-phosphorylation

• Growth induced through MAP kinase and PI3-K pathways

• GLUT4 glucose transporter expression increased

How was insulin produced in Ec using recombinant DNA

A and B chains were expressed separately and refolded into mature insulin. The chains are bound to Beta-galactosidase and purified