Protein Expression in E. coli Flashcards
Why did a journal claim that genes shown to have potentially significant implications in human disease are being ignored due to historical bias?
Previous studies have reported that research is focused around only 2000 coding genes, out of a possible 20,000 genes in the human genome
“Studies on less than ____ of genes comprise more than ____ of research papers and approximately ____ of genes have not been studied at all”
- 10%
- 90%
- 30%
Recombinant protein expression in E.coli: 1966
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
Recombinant protein expression in E.coli: 1967
The first DNA ligase is isolated
Recombinant protein expression in E.coli: 1969
Shapiro and Bechwith isolate the first gene
Recombinant protein expression in E.coli: 1970
The first restriction enzyme is isolated
Recombinant protein expression in E.coli: 1972
Paul Berg creates the first recombinant DNA molecules
Recombinant protein expression in E.coli: 1973
Cohen and Boyer create first recombinant organisms
Recombinant protein expression in E.coli: 1977
Fred Sanger invents a method for sequencing DNA, which later enables researchers to map genomes
What produces milligram quantities of the protein of interest from a cloned gene?
Directed synthesis
What biochemical experiments are an expressed and purified protein used for?
- Activity assays (kinetic and structure/function)
- Antibody production
- Three-dimensional structure determination
- Pharmaceutical purposes
How is E.coli in recombinant protein expressed?
By IPTG induction
What did activity studies of PTEN demonstrate?
PTEN can directly dephosphorylate Pdtlns(3,4,5)P3
What was elucidated from the three-dimensional structure of PTEN?
- Determine functional architecture
- Provide functional clues
- Elucidate molecular details of catalysis
What does the crystal structure of the PTEN tumour suppressor implicate?
Its phosphoinositide phosphatase activity and membrane association
What does the direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveal?
A link between the PAR/aPKC complex and phosphoinositide signaling
How does E.coli grow in rich media?
Rapidly, doubling every 30 minutes at 37 degrees C
What happens if E. coli cells are too dense?
They will deplete the nutrients critical for growth and stop growing - they enter the stationary phase
What are the cell conditions when protein expression experiments are being performed?
The cells are in a rapidly dividing phase - log phase
What are the features of a 6000 bp E. coli vector/plasmid?
- Promoter
- Shine-Delgarno
- N-terminal tag
- Fusion protein
- Protease cleavage site
- Multiple cloning site
- C-terminal tag
- Transcription terminator
- Repressor
- Origin
- Selectable marker
What does the lac operon (gene cluster) of E. coli permit?
Controlled gene expression via chemical induction
How is the lac operon regulated?
Negatively regulated by the transcriptional repressor Lac L in the absence of lactose
What does Lac L do in the presence of lactose or analogues of lactose (i.e., IPTG)?
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
Where is the lac operon promoter commonly engineered in plasmids?
Upstream of your gene of interest
When can the gene of interest be expressed?
When IPTG is added to the mixture
Why is IPTG a strong inducer?
Because it is non-metabolizable
What are the disadvantages of E. coli systems?
- Post-transcriptional modifications
- Protein folding
- Codon usage
Why are post-translational modifications a disadvantage of E. coli systems and what is the solution?
E. coli often lacks sophisticated protein modification systems required for some regulatory enzymes
- In vitro modify purified proteins
Why is protein folding a disadvantage of E.coli systems and what is the solution?
Many eukaryotic proteins are not able to be properly folded in bacterial cells
- Add molecular chaperones to assist in folding
Why is codon usage a disadvantage of E.coli systems and what is the solution?
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
What is the overview of this lab’s steps?
- Induction at log phase (checkAOD600)
- Centrifugation principles
- Cell lysis by sonication
- Protein concentration by Bradford Assay
What samples should be kept at the end of this lab?
- Total cell pellet
- Supernatant of your sonicated sample
