生物化学教学课件：Protein purification

1、1 Protein purification 2014-09-25 2 Hemoglobin 血红蛋白 /wiki/Hemoglobin Two subunits of 141 residues and two subunits of 146 residues. 3 Myoglobin: O2 storage Hemoglobin: O2 transportation 4 Y = pO2n pO2n + P50n n=1, hyperbolic curve; Single or independent binding; n1, sigmoidal c

2、urve; cooperative binding. Hb + n O2Hb(O2)n Hill coefficient, n, reflects the degree of cooperativity. 5/wiki/Hemoglobin Hemoglobin is an allosteric protein. 6 Something else to know about hemoglobin Hemoglobin binds CO with higher affinity; Sickle-cell anemia is caused by hemo

3、globin mutation E6V. 7 ATP synthase is a highly complex molecular machine 8 9 10 Basic principle of protein purification 11 protein purification techniques Based on physical and chemical characteristics: size charge dialysis, gel filtration chromatography, hydrophobicity Based on biological characte

4、ristics: affinity chromatography ion exchange chromatography hydrophobic interaction chromatography, reverse phase chromatography 12 Protein size Molecular weight: NH2-CH-COOH CH2COOH Mr = 133 amino acid -NH-CH-CO- CH2COOH Mr = 115 residue Average molecular weight of residues = 110. A protein of 22

5、KD about 200 residues cDNA of about 600 bp. the ratio of the mass of a molecule to one-twelfth the mass of carbon-12 (12C). often denoted Mr, dimensionless. Molecular mass: the mass of one molecule, expressed in daltons (Da). 13 Dialysis 透析 Dialysis tube with MWCO; Buffer volume; The times of buffer

6、 change. Dialysis time; 14 Ultrafiltration 超滤 (Centricon from Millipore) 15 Gel filtration chromatography Also known as size exclusion chromatography; Effective separation range (globular proteins); Exclusion limit; Limited sample volume; Bed volume and void volume; Scanning electron micrograph of a

7、n agarose gel. Magnification x 50,000. (Ref. Anders S. Medin,PhD Thesis, Uppsala University 1995) 16 17 18 Example: Superdex 75 from GE Healthcare 19 Ion exchange chromatography (from GE Healthcare) 20 Ion exchange chromatography 21 22 Type of Ion Exchange Common Abbreviation Functional group Strong

8、 AnionQ Quarternary Ammonium CH2N+(CH3)3 Weak AnionDEAEDiethylaminoethyl C2H4N+H(C2H5)2 Strong CationSSulfonic Acid CH2SO3- Weak CationCMCarboxymethyl CH2COO- Ion exchange chromatography 23 Ion exchange chromatography Could be eluted by increasing salt concentration or changing pH condition; Stepwis

9、e elution or gradient elution; 01020304050 0.0 0.2 0.4 0.6 0.8 1.0 Salt (M) Elution (ml) 01020304050 0.0 0.2 0.4 0.6 0.8 1.0 Salt (M) Elution (ml) 24 Example: Mono S column from GE Healthcare Ribonuclease A pI=9.45 Cytochrome C pI=10.0-10.5 Lysozyme pI=11.35 25 Hydrophobic interaction chromatography

10、 (HIC) Hydrophobicity: octyl phenyl butyl (http:/www.chisso.co.jp/fine/en/cellufine/images/hyd_partial.gif) (from GE Healthcare) 26 Protein solubility and pH condition When pH pI- When pH pI0 0 0 0 Avoid pH pI to better solubilize proteins! 27 Protein solubility and salt condition salting-in effect

11、salting-out effect 28 Affinity chromatography Flash 29 Immobilized metal chelating chromatography (IMAC) Popularly used for (His)6-tagged proteins. Imidazole to elute; More options of some other metal ions (Co2+, Cu2+); (from Qiagen) 30 Commonly used affinity strategies for recombinant proteins Fusi

12、on Tag Immobilized Ligand Elution Conditions Glutathione S- transferase(GST) Reduced glutathione Free reduced glutathione at neutral pH (competitor) Histidine-tagged Chelated Nickel or Cobalt 200 mM Imidazole, low pH, or strong chelators Maltose Binding Protein (MBP) DextrinMaltose at neutral pH (competitor) 31 Change condition to elute NoYesYesYesYes Sample vol