Question

4. You are investigating the effect of glycosylation on the binding of the Angiotensin- converting enzyme-2 (ACE-2) protein with the SARS-CoV2 virus spike protein. The ACE-2 protein has 7 N-glycosylation sites and 1 O-glycosylation site. Two N-glycosylation sites (Asn- 90 and Asn-322) have been shown to be of particular importance in viral binding. Presence of the N-glycan at Asn-90 interferes with viral binding, while the N-glycan at Asn-322 enhances binding of the ACE-2 receptor binding domain of the SARS-CoV2 spike protein. You have been provided with human cell lysate containing the native ACE-2 protein. You need to purify this protein for antibody production and structural analysis by x- crystallography to enable future drug design. The native, human ACE-2 protein has a molecular weight of approx. 90.7-92.5 kilodaltons (kDa), and a theoretical isoelectric point (pl) of 5.36. (a) Describe the workflow that you would use to purify the native protein to the desired purity, explaining the principles of the bioseparation methods you would choose, and why you have selected these methods (70 marks). (b) Briefly outline how you would check the glycosylation of your purified ACE-2 protein (30 marks). PTO

          4. You are investigating the effect of glycosylation on the binding of the Angiotensin-
converting enzyme-2 (ACE-2) protein with the SARS-CoV2 virus spike protein. The ACE-2
protein has 7 N-glycosylation sites and 1 O-glycosylation site. Two N-glycosylation sites (Asn-
90 and Asn-322) have been shown to be of particular importance in viral binding. Presence
of the N-glycan at Asn-90 interferes with viral binding, while the N-glycan at Asn-322
enhances binding of the ACE-2 receptor binding domain of the SARS-CoV2 spike protein.
You have been provided with human cell lysate containing the native ACE-2 protein. You
need to purify this protein for antibody production and structural analysis by x-
crystallography to enable future drug design. The native, human ACE-2 protein has a
molecular weight of approx. 90.7-92.5 kilodaltons (kDa), and a theoretical isoelectric point
(pl) of 5.36.
(a) Describe the workflow that you would use to purify the native protein to the desired
purity, explaining the principles of the bioseparation methods you would choose,
and why you have selected these methods (70 marks).
(b) Briefly outline how you would check the glycosylation of your purified ACE-2 protein
(30 marks).
PTO

4. You are investigating the effect of glycosylation on the binding of the Angiotensin-
converting enzyme-2 (ACE-2) protein with the SARS-CoV2 virus spike protein. The ACE-2
protein has 7 N-glycosylation sites and 1 O-glycosylation site. Two N-glycosylation sites (Asn-
90 and Asn-322) have been shown to be of particular importance in viral binding. Presence
of the N-glycan at Asn-90 interferes with viral binding, while the N-glycan at Asn-322
enhances binding of the ACE-2 receptor binding domain of the SARS-CoV2 spike protein.
You have been provided with human cell lysate containing the native ACE-2 protein. You
need to purify this protein for antibody production and structural analysis by x-
crystallography to enable future drug design. The native, human ACE-2 protein has a
molecular weight of approx. 90.7-92.5 kilodaltons (kDa), and a theoretical isoelectric point
(pl) of 5.36.
(a) Describe the workflow that you would use to purify the native protein to the desired
purity, explaining the principles of the bioseparation methods you would choose,
and why you have selected these methods (70 marks).
(b) Briefly outline how you would check the glycosylation of your purified ACE-2 protein
(30 marks).
PTO

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