1、Protein Purification Strategies,Course: Methods in protein chemistry Rahman M. Mahfuzur 2012/01/11 SLU,To seperate a particular protin from all other proteins and cell components There are many types of proteins within an ogranism Other components: nuclic acids, charbohydrates, lopids, small molecul
2、es A gieven protein could be 0.001-20% of total protein.,Objectives,Proteins purification varies from one purification step to multi- step of purifixcations Often more than one purification step is necessary to reach the desired purity, or step can be repetead if sample is available. Successful and
3、efficient protein purification depends on appropriate methods selection. Methods should be sequence in a logical manner,what kinds of materials are available/handle? what has to be removed/ completely? what will be the use of final products? what are economical constraints?,Protein purification,Thre
4、e phase purification strategies(CIPP),The four parameters,Every technique offers a balance between resolution, capacity, speed and recovery,Capture,Initial purification of target Rapid isolation, stabilization, concentration,Intermediate purification,Further removal of bulk contaminants: other prote
5、ins, nucleic acids, endotoxinsand viruses. Purification and concentration.,Polishing,Final removal of remaining trace impurities or closely related substances to achieve high purity,Protein properties Vs Technique,Ion exchange chromatography (IEX),separates proteins with differences in surface charg
6、e. based on the reversible interaction, charged protein Vs oppositely charged column If, pHbPIP Neg. bind positively charged anion exchanger ; ex- MonoQ column When, pHbPIP Pos. bind negatively charged cation exchanger ; ex- MonoS column,IEX chromatogram,Affinity chromatography (AC),On the basis of
7、a reversible or specific interaction between target and a specifc ligand Biospecific: antibodies binding proteins, Non-biospecific: histidine binding protein bind to metal Ion; IMAC,AC chromatogram,Gel filtration chromatography (GF),allow separation of proteins with differences in molecular size GF
8、is a non-binding method 0.5% to 2% of total column volume.,FG chromatogram,Hydrophobic interaction chromatography (HIC),separates proteins with differences in hydrophobicitybased on the reversible interaction between a protein and the hydrophobic surface of a chromatography medium,Technique Vs three phase,combinations of chromatographic steps,IEX-HIC-GF,Considered as a standard protocol If nothing is known about the target protein use IEX-HIC-GF. both anion and cation exchange could be used to get different selectivities within the strategy.,Thanks for listening me!,
