Protein Purification

Question 1

Specific activity

Answer 1

= Total activity (Units) / Total protein (mg)

Question 2

Extraction by Homogenization

Answer 2

• By grinding the tissues – the simplest approach, it breaks also the subcellular organelles, such as mitochondria, peroxisomes and endoplasmic reticulum;
• Potter–Elvejhem homogenizer, a thick-walled test tube through which a tight-fitting plunger is passed. It leaves many organelles unopen;
• By freezing and thawing cycles (technically simple and mild);
• Sonication – using ultrasound waves to break open the cells (time
of sonication is adjustable);
• Use of detergents is needed when proteins are bound to the cell
wall;

Question 3

Filtering by Differential Centrifugation

Answer 3

i. First spinning the sample at 600 × g
- a pellet of unbroken cells and nuclei;
ii. Supernatant centrifuged at 15,000 × g
- brings down the mitochondria;
iii. Further centrifugation at 100,000 × g
- brings down the microsomal fraction, consisting of ribosomes and membrane fragments

Question 4

Column Chromatography

Answer 4

 Column chromatography – refers to several common techniques for purification of proteins.
 Gel filtration (size exclusion) chromatography – proteins are separated by size. Another term – molecular sieve chromatography.
 Ion-exchange chromatography - molecules with a specific charge are selectively bound to a column, separated from proteins that don’t bind, and then eluted.
 Affinity chromatography – molecules are bound to the column via specific interactions for a bound ligand. Once non-bound proteins are removed, the protein of interest can be eluted.

Question 5

Electrophoresis

Answer 5

 Proteins are separated on the gel based on their size, shape, and charge.
 With SDS–polyacrylamide-gel electrophoresis, proteins are separated based on molecular weight.
 With isoelectric focusing electrophoresis, proteins are separated based on their isoelectric points.

Question 6

Gel-Filtration Chromatography

Answer 6

Advantages:
1. Convenience;
2. Ability to estimate molecular weights of eluted substances by using the samples with a set of standards;
Disadvantages:
1. Each type of gel used has a specific rather narrow range of sizes to be separated:
2. Overlaps between eluted proteins;

Question 7

Ion-Exchange Chromatography

Answer 7

Advantage: Generally applicable;
Disadvantage:
Low level of purification.

Question 8

Affinity Chromatography

Answer 8

Advantages:
High level of purification of the desired protein;
Disadvantage:
Each type of affinity columns targets only one group of specific proteins.

Question 9

Edman degradation method.

Answer 9

Identification of protein sequences by cleavage at specific amino acid residues to short peptides

Question 10

Trypsin Cleavage

Answer 10

The cleavage takes place at basic amino acids arginine and lysine in such a way that the amino acid with the charged side chain ends up at the C-terminal end of one of the peptides produced by the reaction except when followed by proline

Question 11

Chemotrypsin Cleavage

Answer 11

Cleaves peptide bonds preferentially at the aromatic amino acids: tyrosine, tryptophan and phenylalanine. The aromatic amino acid ends up at the C-terminal ends of the peptides produced by the reaction.

Question 12

Cyanogen Bromide Cleavage

Answer 12

In the case of the chemical reagent cyanogen bromide (CNBr), the sites of cleavage are at internal methionine residues. The sulphur of the methionine reacts with the carbon of the cyanogen bromide to produce a homoserine lactone at the C-terminal end of the fragment.