Pemurnian Protein [PDF]

Citation preview

TEKNIK ISOLASI PROTEIN Protein ekstraseluler  Tahapan isolasi 



Sentrifugasi cairan sel/ medium kultur ⇒ menghasilkan :  Supernatan (crude extract)  Pelet (sel & komponen non-protein)



Protein intraseluler  Tahapan isolasi : 











Cuci jaringan/ sel dengan bufer salin : untuk menghilangkan pengotor/ bahan ekstraseluler  Sel mikroba : sentrifugasi & resuspensi dalam bufer Lisis sel ⇒ memecah/membuka sel ⇒ ekstrak : homogenat  Menggunakan mortar/pestle, homogenizer, sonicator, tissue grinder, cell disruptor, blender    Menghasilkan : homogenat Sentrifugasi ⇒ menghasilkan :  pelet (mengandung protein membran)  supernatan (crude extract)



Cell disruption methods Cell lysis method



Kind of tissue



Blade homogenization



Most animal, plant tissue



Hand homogenization



Soft animal tissue



Sonication



Cell suspention



French pressure cell



Bacteria, yeast, plant cell



Grinding with alumina/ sand



Bacteria, plant cell



Glass bead vortexing



Cell suspention



Enzyme digestion



Bacteria, yeast



Detergent lysis



Tissue culture cell



Organic solven lysis



Bacteria, yeast, plant cell



Osmotic shock



Erythrocytes, bacteria



Removal of Non-Protein Components



PROTEIN SEPARATION The goal of a protein separation is to



obtain the protein in a pure, active form using a  minimum number of steps The shortest time possible.



SEPARATION METHOD  Chromatographic Methods Proteins differ in size and charge Ion exchange chromatography Anion and Cation



Gel Filtration (size exclusion) Separates based on size Affinity Methods Affinity for ligands Engineered affinity sites, e.g. histidine tag



Chromatography



Solubility techniques  Salting out methods  Solubility sensitive to ionic strength. Initial “salting in” and then“salting out” At high salt, solvent tied up with interacting with salts so that it is insufficient to solubilize proteins.  1st go to maximal salt that protein target is soluble, centrifuge and discard pellet. Now add just enough salt to bring down protein. Collect pellet.  Organic solvents  Same principle as salting out; taking advantage of different solubilities. Avoid totally denaturing proteins.  pH  Proteins have many ionizable groups with range of pKs. When net charge of protein is zero, this is the isoelectric point or pI. Proteins are typically least soluble at their pI due to minimizing charge charge interactions.  Crystallization  The solubility methods where proteins are ppt can be used to grow crystals of proteins. This is only done when the protein is relatively pure



Ammonium sulfate precipitation (salting out)  When high concentration of salt are present, proteins



tend to aggregate and precipitate out of solution : “salting out”  Different proteins precipitate at different salt concentration. pH, temperature and protein purity play important roles in determining the salting out poin.  Ammonium sulfate is the salt choice because it combines many useful feature :     



salting out effectiveness pH versality high solubility low heat of solution low price



 Ammonium sulfate concentration : % saturation  Simple equation for calculation of gram ammonium sulfate needed



to make an X% solution from Xo% : 515 (X-Xo) g = ------------100-0,27X



Dialys is



ION EXCHANGE CHROMATOGRAPHY Low salt P+



+ Na+



Na+ + P+ High salt



IONIC ELUTION pH ELUTION



Ion exchange groups used in protein purification



─SO3-



STRONG CATION ─ Sulpho



STRONG ANION ─ CH2N+(CH3)3 ─ Triethylaminomethyl



─ CH2SO3-



─ Sulphomethyl



─ C2H4N+(C2H5)3



─ C3H6SO3-



─ Sulphopropyl



─ C2H4N+(C2H5)2CH2CH(OH)CH3 ─ Diethyl2-hydroxypropylaminoethyl WEAK ANION ─ C2H4N+H3 ─ Aminoethyl



─ COO-



WEAK CATION ─ Carboxy



─ CH2COO-



─ Carboxymethyl



─ Triethylaminoethyl



─ C2H4N+H(C2H5)2 Diethylaminoethyl



─



Gel Filtration  Porous beads made of different



materials. Size of pores can be controlled  Small molecules small enough to go into beads whereas larger go around



and thus flow faster. There is exclusion limit (all proteins too large to go into pores).  Can be used as preparative method



or be used to determine molecular size  Gels made of dextrans, agarose or



Affinity Method A ligand which has tight



binding to protein is attached to matrix.  Protein of interest binds but



others pass through  Elute using soluble ligand.  Beside small molecules, can also use antibodies  Recent advances in molecular biology entails engineering a poly his group which binds to Ni or use parts of other proteins and columns that bind to this other protein



Addition of glucose (G)



Visualization  &/or  Isolation  of a protein   proteins migrate in an electrical field at rates



that depend upon their net charge, size, and shape  Gel Electrophoresis... separation by charge ( separation of in a media as gels)  in a media as porous gel (starch/polyacrylamide) : gels & staining  Isoelectric Focusing*: proteins are separated in a gel of a continuous pH gradient, proteins move to point in gel equal to its pI, i.e., no charge  SDS-PAGE*: Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis... proteins treated with ionic detergent that separates according to size  SDS binds to protein @ 1 SDS/2 aa's thus proportional to a protein's MW           



SDS-PAGE



Identification of protein’s presence & &  its quantification    Identification - is often done by  spectrophotometry    



     spectrophotometers measures intensity of light beam before & after        light passes through a liquid solvent with sample dissolved in it,         (in a cuvette)... compares the two light intensities over a range of        wavelengths.                           Percent transmittance...       ratio of intensity of light passing through the sample       to the intensity of light shining on sample multiplied by 100%.   Absorbance...         is the log of the transmittance                                instruments... Spectronic 20/  spectrophotometer UV/ Vis  



SPECTROPHOTOMETRIC  METHODS  of  DETECTING  PROTEINS UV absorbance at 280 nm. (measures aromatic aa's)        Colorimetry reactions - colored dye binds to amino acids           Ninhydrin reaction - rx's w amino = blue color (10-9 M)                  Biuret test = mg quantities…   based on Copper ion                                 binds stiochiometrically = violet color            Bradford test = ug amounts            based on dye Coomassie blue - binds to peptide              Fluoroescamine dye = pg quantities...  (10-12 M) Quantification of amounts of protein present  Quantification is based on BEER-LAMBERT Law            



Quantification  of Protein Concentrations with ENZYME ACTIVITY relating protein amounts & enzyme activity



           1  (international)  UNIT of  ENZYME ACTIVITY…          that amount of protein which converts 1 micromole of substrate to product per min at 250C at optimal pH UREASE - 1 unit (IU) will liberate 1.0 µmole of ammonia from         urea per minute at pH 7.0 at 25°C    [equivalent  to 1.0  I.U.]               1  UNIT of  SPECIFIC ACTIVITY…            the number micromoles converted per min  per mg protein            i.e., Units (as above) of enzyme activity per mg



Protein Purification