Pemurnian Protein

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Transcript of Pemurnian Protein

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TEKNIK ISOLASI PROTEIN Protein ekstraseluler

Tahapan isolasi Sentrifugasi cairan sel/ medium kultur menghasilkan :

Supernatan (crude extract) Pelet (sel & komponen non-protein)

Protein intraselulerTahapan 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)

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Cell disruption methodsCell 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

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Removal of Non-Protein Components

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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.

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SEPARATION METHODChromatographic Methods

Proteins differ in size and chargeIon exchange chromatography

Anion and CationGel Filtration (size exclusion)

Separates based on sizeAffinity Methods

Affinity for ligandsEngineered affinity sites, e.g. histidine tag

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Chromatography

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Solubility techniquesSalting 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

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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

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Dialysis

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ION EXCHANGE CHROMATOGRAPHYLow salt

High salt

P+ + Na+Na+ + P+

IONIC IONIC ELUTIONELUTION pH pH ELUTIONELUTION

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Ion exchange groups used in protein purification

STRONG CATION ─SO3

- ─ Sulpho

─ CH2SO3- ─ Sulphomethyl

─ C3H6SO3- ─ Sulphopropyl

WEAK CATION ─ COO- ─ Carboxy

─ CH2COO- ─ Carboxymethyl

STRONG ANION ─ CH2N+(CH3)3 ─ Triethylaminomethyl

─ C2H4N+(C2H5)3 ─ Triethylaminoethyl

─ C2H4N+(C2H5)2CH2CH(OH)CH3 ─ Diethyl-2-hydroxy- propylaminoethyl

WEAK ANION ─ C2H4N+H3 ─ Aminoethyl

─ C2H4N+H(C2H5)2 ─ Diethylaminoethyl

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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 sizeGels made of dextrans, agarose or

polyacrylamide

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Affinity MethodA ligand which has tight

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

others pass throughElute using soluble ligand.Beside small molecules, can

also use antibodiesRecent 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

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Addition of glucose (G)

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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          

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SDS-PAGE

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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  

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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          linear relationship between... light Absorbance vs. Conc   Protein Standard Curve

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Quantification of Protein Concentrations with ENZYME ACTIVITYrelating 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                 1  UNIT of  MOLECULAR ACTIVITY…              number of units of enzyme activity per µmole

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Protein Purification