V. Lab | 75. Purification of a bacterially expressed protein by affinity chromatography

Question 1

I. Background information
1. What is recombinant protein production?

Answer 1

• An artificial DNA sequence (recombinant DNA) will be inserted into an appropriate protein overexpression vectors.
• The vectors are delivered into prokaryotic/eukaryotic host cells which can be induced to express the recombinant protein.
• Before use, the target protein must be separated from the useless but abundant endogenous proteins of the host cell.
• Protein purification is performed with a special chromatography assay such as affinity chromatography -> a tool used to achieve a high- grade purification in a single step.
• Accordingly, recombinant proteins are often tagged with short amino acid sequences that make the application of affinity chromatography possible.

Question 2

II. Experiment
1. What is the aim of Purification of a bacterially expressed protein by affinity chromatography

Answer 2

• In this lab, the aim is to purify the yellow fluorescent protein (YFP) expressed in E.coli bacterial cells.
• YFP is tagged with six contiguous histidine residues (6xHis) at its N-terminal, enabling its affinity chromatography-based purification.

Question 3

II. Experiment
2. How can successful induction be detected?

Answer 3

• Successful induction can be detected by illuminating the cell suspensions with UV light.

Question 4

II. Experiment
3. What happen after successful induction?

Answer 4

• Following induction, the bacterial cells can be separated from their culture broth with a low- speed centrifugation step.
• The centrifugation results in a bacterial pellet at the bottom of the tube and a liquid supernatant above.
• By examining both phases under UV light, we can observe that the induced bacterial cell pellet emits the most intensive fluorescence.

Question 5

II. Experiment - Cell lysis
4A. What are the main components of lysis solution?

Answer 5

• The main components of the lysis solution are as follows: phosphate buffer, sodium chloride, lysozyme, DNase

Question 6

II. Experiment - Cell lysis
4B. What is the role of phosphate buffer in the lysis solution?

Answer 6

Phosphate buffer ensures the optimal pH for lysis (pH = 8)

Question 7

II. Experiment - Cell lysis
4C. What is the role of NaCl in the lysis solution?

Answer 7

NaCl provides appropriate ionic strength

Question 8

II. Experiment - Cell lysis
4D. What is the role of lysozyme and DNase enzymes in the lysis solution?

Answer 8

lysozyme and DNase enzymes promote cell lysis

Question 9

II. Experiment - Cell lysis
4E. What happen if there is Disruption of cell wall + membrane?

Answer 9

Disruption of cell wall + membrane causes chromosomal DNA to be released
-> greatly increases the viscosity of the suspension
-> DNase added to the lysis solution will degrade all forms of DNA + RNA => decreasing solution viscosity

Question 10

II. Experiment - Cell lysis
4F. What happen after resuspension?

Answer 10

• Following resuspension, cell lysates are to be spinned down at maximal rpm to sediment the larger bacterial debris (e.g. cell membrane, cell wall fragments) and insoluble particles to the bottom of the tube, while the soluble proteins (6xHis-YFP) will remain in the supernatant
• At this step, the supernatant of the induced samples has higher fluorescence under UV. The supernatant (lysate) of the induced sample will be used for affinity chromatography purification