As a reminder your answers must byorwn Please be sure all an

As a reminder, your answers must byorwn Please be sure all answers are in your owo words and to cit all your sources for each question. in your own words and to cite all L. A fasomating new protease in the hindgut of a fnuit tly may be the cure for the common cold it you can isolate, purify, and determine the specific determinants of the enzymatic activity You also know the pl of this protein that it uses copper, and selectively cleaves afer N^-linked glycosylation sites Descrive a method for isolating this proterin. What conditions you would use to test the enzyme activity? HYou\'re initial purified enzyme preparation appears to have itle to ho activ/ity, though you\'ve yerifea by tendem MS that your protein is present. Whataditional factors mightyouneed t) consder\' Bonus points will be given if you incorporate a way to measure the enzyme activity into this answer. MacBook Pro 2.0 12.

Solution

the characteristics  about the protein are:

-Its ability to bind to copper (copper will be the metal cofactor for the protein).

-The pI of the protein

-It cleaves after N-linked glycosylation sites. (glycan chains bound to the N of Asparagine in the sequence Asn- X –Ser/ Thr)

we need to isolate and purify the protein, using these characteristics,

FOR PURIFICATION:

Initially an isoelectric focusing can be done, (a gel where proteins segregate depending on the pI)- so that the proteins having the specific pI can be separated.

more than one protein (including our protein of interest) can have the same pI. So all the protein present in that pI range are taken and then subjected to affinity chromatography where the column of the chromatography will contain copper ions.

Our protein of interest is supposed to bind and stick to the column while the others are to pass through. Then we can elute the protein by adding an elution buffer and obtain the protein of interest.

FOR ENZYME ACTIVITY MEASUREMENT:

Enzyme activity is defined as the number of moles of substrate converted to product per unit time. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified.

So the assay should be conducted in a pH corresponding to the pI of the enzyme and the substrate should be N-linked glycosylated proteins where the glycosylated chains may be tagged. Also in the buffer, copper must be present as it is presumably an activator for the protein.

Failure to incorporate any one of the above-mentioned steps might lead to the inactivity of the enzyme and hence, the enzyme activity cannot be assayed.