Protein purification

Sources of protein purification

• sperm whale myoglobin

• prokaryotes

• unicellular eukaryotes

• mammalian cells

adv + disadvantage of prokaryotic cell as source

ADV:

• very easy to grow in large quantities

• easy to genetically manipulate

DISADV:

• post transitional modifications are very different

• complicated protein shapes can’t be formed 

adv + disadvantage of eukaryotic celll as source

ADV:

• high yield 

• post transitional modifications are similar to those in mammals 

DISADV:

• limited ability to fold and form complicated proteins 

adv + disadvantage of mammalian cellsl as source

ADV:

• can fold proteins like humans 

• full range of post transitional modifications

DISADV

• hard to genetically manipulate 

• low yields 

describe ion exchange

separating proteins in a column according to its protein charge;

• choose IEX media according to protein net electrical charge . Below pI, protein is positive and above pI protein is negative

• load protein mixture in buffer with low salt concentration

• wash column with low salt buffer to remover unbound proteins

• elute proteins by increasing salt concentration of buffer (proteins with weaker interactions elute first)

describe gel filtration chromatography

separating proteins according to its protein size :

• column is packed with porous beads of specific size

• protein sample dissolved and passed through column in buffer solution 

• larger proteins are excluded and travel around beads

• proteins smaller than pores enter bead, causing them to elute later

how can protein purity be determined 

using SDS-page combined with an activity assay:

• acrylamide gel which acts as a sieve 

• SDS and heat treated proteins applied to gel

• SDS gives proteins a negative charge 

• voltage applied to gel, causing proteins to migrate towards positive electrode 

• lower molecular weight runs faster than higher molecular weight