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 results 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 cDNA.
(a) What type of vector should the researcher use? Describe the elements this vector should contain and identify the sites required to allow for digestion, ligation, regulation, transcription and translation.
(b) Identify a commercially available vector that you would suggest the researcher employ in his/her research and support this suggestion with reasoning. (You can make assumptions as needed as long as they are disclosed).
(c) Provide the specific enzymes and reagents required to over-express this protein using recombinant DNA generated from the vector you have suggested. Detail each technique required to undergo this process from gene to protein. Make sure to highlight the biochemistry that facilitates each step.
(d) Would you expect the protein produced by E. coli to function similarly to that isolated from mammalian cells? Provide an explanation.
(e) Explain why the researcher depends on the preparation of cDNA to define gene or protein function.
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 results 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 cDNA.
(a) What type of vector should the researcher use? Describe the elements this vector should contain and identify the sites required to allow for digestion, ligation, regulation, transcription and translation.
(b) Identify a commercially available vector that you would suggest the researcher employ in his/her research and support this suggestion with reasoning. (You can make assumptions as needed as long as they are disclosed).
(c) Provide the specific enzymes and reagents required to over-express this protein using recombinant DNA generated from the vector you have suggested. Detail each technique required to undergo this process from gene to protein. Make sure to highlight the biochemistry that facilitates each step.
(d) Would you expect the protein produced by E. coli to function similarly to that isolated from mammalian cells? Provide an explanation.
(e) Explain why the researcher depends on the preparation of cDNA to define gene or protein function.
