2 A biochemist would like to use E coli to produce a mammali

2. A biochemist would like to use E. coli to produce a mammalian protein. The protein is 40 kDa in size with approximately 20% of its mass resulting from polysaccharides. It was found, during previous experimentation, that when isolating the glycoprotein, it was phosphorylated and formed four distinct disulfide bonds. The cloned gene the researcher has been using was prepared through the construction of a cDNA. (a) what type of vector should the researcher use? Describe the vector and identify the sequences and sites required to allow for digestion, ligation, regulation, transcription and translation. (b) Identify a commercially available vector that you would suggest the researcher use and support this suggestion with your reasoning. (You can make assumptions as needed as long as they are disclosed). on Slides new engian d bio u abs

Solution

First of the size of protein is too large that is 40kb, for that a specilaized fosmid molecule has to inserted to increase E.Coli capacity. Most probably the protein will be truncated in basic E.Coli

Answer d.) E.Coli protein will not be functionally similaar to mammalian cell because E. Coli lacks chaperones which provide proper folding to protein, unique specialised post-translational modifications which direct protein localization in sub-cellular and its function and it dont posses support system to form disulphide bond.

To troubleshoot such problem, co-expression of chaperons and post translational enzymes are induced within the system (either heat shock or chemical method) or use genitically modified E Coli strains that have chaperone in their genome.