Protein Expression in E. coli

Question 1

Why did a journal claim that genes shown to have potentially significant implications in human disease are being ignored due to historical bias?

Answer 1

Previous studies have reported that research is focused around only 2000 coding genes, out of a possible 20,000 genes in the human genome

Question 2

“Studies on less than ____ of genes comprise more than ____ of research papers and approximately ____ of genes have not been studied at all”

Answer 2

• 10%
• 90%
• 30%

Question 3

Recombinant protein expression in E.coli: 1966

Answer 3

Khorana, Nirenberg, Matthaei, and Ochoa identify which codon sequences indicate each of the 20 amino acids, thereby “cracking the genetic code” and enabling advances in genetic engineering

Question 4

Recombinant protein expression in E.coli: 1967

Answer 4

The first DNA ligase is isolated

Question 5

Recombinant protein expression in E.coli: 1969

Answer 5

Shapiro and Bechwith isolate the first gene

Question 6

Recombinant protein expression in E.coli: 1970

Answer 6

The first restriction enzyme is isolated

Question 7

Recombinant protein expression in E.coli: 1972

Answer 7

Paul Berg creates the first recombinant DNA molecules

Question 8

Recombinant protein expression in E.coli: 1973

Answer 8

Cohen and Boyer create first recombinant organisms

Question 9

Recombinant protein expression in E.coli: 1977

Answer 9

Fred Sanger invents a method for sequencing DNA, which later enables researchers to map genomes

Question 10

What produces milligram quantities of the protein of interest from a cloned gene?

Answer 10

Directed synthesis

Question 11

What biochemical experiments are an expressed and purified protein used for?

Answer 11

• Activity assays (kinetic and structure/function)
• Antibody production
• Three-dimensional structure determination
• Pharmaceutical purposes

Question 12

How is E.coli in recombinant protein expressed?

Answer 12

By IPTG induction

Question 13

What did activity studies of PTEN demonstrate?

Answer 13

PTEN can directly dephosphorylate Pdtlns(3,4,5)P3

Question 14

What was elucidated from the three-dimensional structure of PTEN?

Answer 14

• Determine functional architecture
• Provide functional clues
• Elucidate molecular details of catalysis

Question 15

What does the crystal structure of the PTEN tumour suppressor implicate?

Answer 15

Its phosphoinositide phosphatase activity and membrane association

Question 16

What does the direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveal?

Answer 16

A link between the PAR/aPKC complex and phosphoinositide signaling

Question 17

How does E.coli grow in rich media?

Answer 17

Rapidly, doubling every 30 minutes at 37 degrees C

Question 18

What happens if E. coli cells are too dense?

Answer 18

They will deplete the nutrients critical for growth and stop growing - they enter the stationary phase

Question 19

What are the cell conditions when protein expression experiments are being performed?

Answer 19

The cells are in a rapidly dividing phase - log phase

Question 20

What are the features of a 6000 bp E. coli vector/plasmid?

Answer 20

• Promoter
• Shine-Delgarno
• N-terminal tag
• Fusion protein
• Protease cleavage site
• Multiple cloning site
• C-terminal tag
• Transcription terminator
• Repressor
• Origin
• Selectable marker

Question 21

What does the lac operon (gene cluster) of E. coli permit?

Answer 21

Controlled gene expression via chemical induction

Question 22

How is the lac operon regulated?

Answer 22

Negatively regulated by the transcriptional repressor Lac L in the absence of lactose

Question 23

What does Lac L do in the presence of lactose or analogues of lactose (i.e., IPTG)?

Answer 23

Binds to lactose (or IPTG) and a conformation change is triggered that decreases the DNA binding activity of Lac I (and thus eliminates the transcriptional repression), permitting gene expression of the enzyme system

Question 24

Where is the lac operon promoter commonly engineered in plasmids?

Answer 24

Upstream of your gene of interest

Question 25

When can the gene of interest be expressed?

Answer 25

When IPTG is added to the mixture

Question 26

Why is IPTG a strong inducer?

Answer 26

Because it is non-metabolizable

Question 27

What are the disadvantages of E. coli systems?

Answer 27

• Post-transcriptional modifications
• Protein folding
• Codon usage

Question 28

Why are post-translational modifications a disadvantage of E. coli systems and what is the solution?

Answer 28

E. coli often lacks sophisticated protein modification systems required for some regulatory enzymes
- In vitro modify purified proteins

Question 29

Why is protein folding a disadvantage of E.coli systems and what is the solution?

Answer 29

Many eukaryotic proteins are not able to be properly folded in bacterial cells
- Add molecular chaperones to assist in folding

Question 30

Why is codon usage a disadvantage of E.coli systems and what is the solution?

Answer 30

E. coli has less tRNAs for certain amino acids that are prevalent in higher eukaryotic proteins
- Use genetically modified codon, plus E. coli cells, which express extra tRNAs to improve expression

Question 31

What is the overview of this lab’s steps?

Answer 31

• Induction at log phase (checkAOD600)
• Centrifugation principles
• Cell lysis by sonication
• Protein concentration by Bradford Assay

Question 32

What samples should be kept at the end of this lab?

Answer 32

• Total cell pellet
• Supernatant of your sonicated sample