Bacterial Protein Expression Flashcards
Describe how chymosin is now expressed by E. Coli for industrial needs.
Chymosin is used to manufacture cheese. It is traditionally produced by rennet derived from calf stomachs. E. Coli were transformed with the gene for the enzyme to produce the enzyme on a large scale with a good quality of enzyme product. Today about 60% of the US’s hard cheeses are made with GM chymosin.
Why is recombinant human growth hormone used instead of purified HGH from other sources?
HGH used to be derived from the pituitary glands of cadavers however this led to some patients developing iatrogenic CJD.
What is required for bacterial protein expression?
You need to know the DNA sequence of the gene of interest.
The gene must be introduced into an expression plasmid.
The expression plasmid must then be introduced into the bacteria.
The recombinant protein must then be purified.
What are the advantages of using E. Coli for expressing proteins?
There is lots of laboratory support: such as kits, plasmids, reagents.
There is a high yield with E. Coli.
The cost of media and reagents is cheap.
E. Coli has fast growth.
It can be grown on both a small and a large scale.
What are the disadvantages of using E.coli?
There are no post translational modifications.
Large proteins may be difficult to express.
Membrane proteins may not fold.
Some proteins may be toxic to E.coli.
Some proteins may be insoluble or improperly folded.
There is a codon usage difference in E.coli.
What is a plasmid?
A plasmid is a small circular piece of DNA that replicates independently from the host cell’s chromosomal DNA.
When using a gene why may the sequence not be transcribed and translated in E.coli?
Because E.coli has a different codon usage frequency. The DNA code is degenerate with many codons coding for the same amino acid. E.coli may not use the same codons as the gene expresses to code for the same amino acids.
What is traditional cloning?
This method starts with the gene of interest being extracted and purified. The gene is then amplified via PCR and cut with restriction enzymes on each flank of the gene. The plasmid is also cut and the two are combined via DNA ligase.
What is ligation dependent cloning?
In this method no ligases or restriction enzymes are used. The method has a high throughput but is expensive. Instead directional TOPO cloning exploits the ligase activity of topoisomerase I by providing an activated linearised vector. The gene is amplified by PCR using primers with 4 bases (CACC) added to the forward primer. The PCR products are then mixed with the TOPO vector.
When transforming the transformed vector into an E.coli strain what strain is desirable?
One with a low protease activity.
Strains with tRNA for rare codons.
Strains that will help with the folding of the protein.
Strains that are suitable for toxic proteins.
What is the BL21 strain?
It is a commonly used E.coli strain. This is because it provides a high level of protein expression as is deficient in two key proteases.
Describe how chemical transformation can be conducted.
E.coli cells are first made competent by incubating in a solution of divalent cations. They are then mixed with the plasmid on ice. They are then heat shocked by transferring to a 42 degree water bath for 1 min. A small volume of media, such as LB, is then added and the cells allowed to grow at 37 degrees for 1 hour. It is then spread onto an agar plate containing the appropriate antibiotics.
After chemical transformation what occurs to the transformed bacteria?
A single colony is taken and grown in media + antibiotics overnight at 37 degrees. After this the growth media is added to a larger volume of media + antibiotics and allowed to grow. Once the OD at 600nm is 0.4-0.6 then the E.coli are ready to start producing the protein.
Once cells are at the right stage of growth how is protein expression induced?
When cells reach mid log phase they can be induced to express the protein. IPTG is added which mimics allolactose and triggers transcription of genes under the control of the lac promoter, it binds to the lab Repressor allowing genes to be transcribed.
How is the protein purified?
After a suitable amount of time growing, the cells are harvested. Media is centrifuged and the cells washed by resuspending in media. They are then broken to release contents. The pellet is discarded after Centrifugation with the protein being in the supernatant.
