Question

parts a and b please

2. In order to analyze a few promising candidates from question 1 in more detail, you will overexpress the mutant versions in bacterial cells for purification via affinity chromatography, a. Describe in bullet points (your own words) how affinity chromatography works. b. Trouble shooting: After affinity chromatography you analyze your fractions by SDS PAGE with Coomassie staining. Only the total lysed cells and the soluble crude lysate contains your enzyme of interest. All other fractions (flow through, washes, and eluates) did NOT contain the enzyme of interest. Describe a scenario why this could have happened and what you would need to do differently next time.

Answer 1

2. a. AFFINITY CHROMATOGRAPHY

• Affinity chromatography principle works on the fact of attachment of the protein or ligand with the particular binding molecule
• If we consider the nickel beads and the histidine tag due to the transition element attachment the bonding between these two molecules will take place
• Considering the golf post and the golf ball, in that particular goal, the only golf ball can fit and not the football thus during the affinity chromatography we use such beads which shows the affinity towards our desired protein and not towards other.
• Thus whenever the protein has been isolated it is very difficult to find the binding molecule every time then we genetically modify that protein in such a way that the protein will have the histidine chain at its one end which can bind to nickel.
• This nickel is present in the matrix and attached to the linker protein.
• The binding molecule can change from nickel to RNA or restriction endonuclease, etc.
• Example if we are using the Ni then the protein will bind in the matrix then to elute out the protein we should use the salt concentration solution which can break those bonds and elute out the protein

Affinity-Tag and Desalting Purification Desalting Affinity Wash Bind Elute Automatic (with Profinia system) Target protein Other components of complex preotein · Salt Ligand Affinity resin Desalting/size exclusion resin mixture

b.The protein or enzyme is not found in the washes, flow throughs or elution and only found in the lysate because it has been purified using the affinity chromatography. Thus, it is the possibility that the protein must have entered into the inclusion bodies which are unable to separate out in the affinity chromatography and thus did not appear on the SDS-PAGE. But in the cell lysate after the cell disruption has been taken place the protein will be observed in the pellet portion and not supernatant. If we have to resolve this problem ahead then the gene of the protein has to be tagged with the signaling gene which will help the protein to release out of the cell and thus able to separate out without disrupting the cells and able to purify easily using the chromatography technique. The genetic design for it will be as follows: