A friend of yours isolated the coding sequence for pretendin from sick individua
ID: 253503 • Letter: A
Question
A friend of yours isolated the coding sequence for pretendin from sick individuals. Now you are trying to express large amounts of the mutated pretendin in bacteria, so that you may be able to purify the mutant protein and run a series of biochemical tests on it trying to figure out what's wrong with it. However, you encounter a problem... Even though you were able to confirm that the DNA for pretendin has been successfully incorporated into the bacterium, no protein is being produced! You check the sequence of the DNA that went into the bacterium, and you notice that it has everything in needs to be expressed in eukaryotic cells. Taking into account what you know about differences in expression between eukaryotes and prokaryotes, what may have gone wrong....? In 150 words or fewer, formulate a hypothesis to explain why the mutant pretendin DNA may not be expressed by the bacteria, and briefly describe an experimental strategy to test your hypothesis. Deadline: Thur 04/05 before class.Explanation / Answer
When we express a eukaryotic gene ( here pretendin) in a prokaryotic system, ( here in bacteria) several problems can happen which will reduce the productivity of of heterologous expression. Such as-
i) Due to difference in the posttranslational modification, the protein which has expressed is not folded properly which can make the expressed protein insoluble. Or if the protein production rate is very high, the host cell may not fold the protein in its actual configuration. As a result we can not identify the protein by SDS-PAGE/ coomasie staining.
ii) In some insances, the promotor and the heterologous gene may not combined well with each other, as secondary structure might form between 5'UTR and begining of the codon. As a result, the ribolome might not translete the mRNA properly.
iii) In some instances, the codon usage between the host cell and the heterlogous protein is not compatible to each other.
iv) Some host bacteria might not suitagble for expressina particluar protein of interest,
to check which one have occured here for the production of mutated pretendin proein we might perform different tests. they are like.
i) After lysing the host cell, while centrifugin we can take the supernatant acnd check the presence of the protein. Also the growth temeprature of bacterium can be slow down to reduce the rate of protein production.
ii) we can try different promtoer , othe than what is there in the bacteria for the expression of pretendin.
iii) For checking codon bias, we can use different bacterial strain like Stragene’s Rosetta of E.coli which have extra t-RNA copies than normal E.coli.
iv) Alternatively for pretendin production different host other than the experimental one can be used.