Protein purification and characterisation

Question 1

What are the advantages of using bacteria to overexpress interesting genes?

Answer 1

• very easy to manipulate
• easy to grow in large quantities
• potentially very high yields of protein

Question 2

What are the disadvantages of using bacteria to overexpress interesting genes?

Answer 2

• post-translational modifications will probably be different to those used in eukaryotic cells
• Poor at folding complicated proteins

Question 3

What are the advantages of using unicellular eukaryotes to overexpress interesting genes?

Answer 3

• easy to genetically manipulate
• easy to grow in large quantities
• potentially high yields of protein
• post-translational modifications more mammalian ones

Question 4

What are the disadvantages of using unicellular organisms to overexpress interesting genes?

Answer 4

• only moderate ability fold complicated proteins

Question 5

What are the advantages of using mammalian cells to overexpress interesting genes?

Answer 5

• Full range of mammalian post-translational modifications
• Ability to fold and assemble complicated proteins

Question 6

What are the disadvantages of using mammlian cells to overexpress interesting genes?

Answer 6

• Less easy to genetically manipulate
• hard to grow in large quantities
• poor yields of protein
• very expensive growth media

Question 7

How can you find out if a protein is present in a sample?

Answer 7

Test for activity by doing an assay

Question 8

What is the most common technique for purifying large quantities of protein?

Answer 8

Column chromatography

Question 9

What is the stationary phase in column chromatography?

Answer 9

The porous solid matrix (the gel)

Question 10

What are the 4 types of column chromatography?

Answer 10

• Gel filtration
• Ion exchange
• Hydrophobic interaction
• Affinity Chromatography

Question 11

What does affinity chromatography separate proteins on?

Answer 11

The basis of specific protein interactions e.g. enzyme binding to a substrate

Question 12

What does gel filtration chromatography separate proteins based on?

Answer 12

The size of the proteins

Question 13

What is the isoelectric point of a protein?

Answer 13

The pH at which a protein has no overall charge

Question 14

What is the charge of a protein at a pH greater than its Pi?

Answer 14

Negative

Question 15

What is the charge of a protein at a pH lower than its Pi?

Answer 15

Positive

Question 16

How can the charge of a protein be changed?

Answer 16

Changing the pH of the buffer

Question 17

Give 2 examples of common ion exchange chromatography media

Answer 17

• DEAE cellulose
• CM cellulose

Question 18

What is the charge of DEAE cellulose media?

Answer 18

Positive

Question 19

What is the charge of CM cellulose media?

Answer 19

Negative

Question 20

What are the 2 ways we can make a protein unbind from a column?

Answer 20

• Change the pH of the buffer flowing through the column to change the charge on the protein
• Increase the buffer [salt] to elute the protein via charge shielding

Question 21

What is used to determine the purity of a protein?

Answer 21

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