Protein purification and characterization

Protein purification

• Purity is defined by the general level of protein contaminants and also by the absence of contaminants of special interests such as microbes, toxins etc.
• Protein purification is divided into five stages:
  1. Preparation of sources
  2. Knowledge of protein properties
  3. Development of an assay
  4. Primary isolation
  5. Final purification

I. Preparation of sources:

• The raw materials from which proteins can be isolated such as microbial culture or animals or plant sources should be selected.
• The amount of protein can be increased by increasing cultivation volume.

II. Knowledge of protein properties:

• Before employing any procedure, one should know about different properties of proteins such as- intracellular or extracellular occurrence, denaturation temperature, pH range, ionic stability, molecular weight, charge, iso-electric point, binding partners etc.

III. Development of an assay:

• An assay developed should be convenient, easy, rapid, precise for purification.

IV. Primary isolation:

• This consists of separation of protein from other cellular components.
• For this propose there are different methods:
• Concentration:
  • Different methods can be employed for concentration of extracellular protein.
  • Ultrafiltration are usually used to concentrate extracellular proteins from cell.
  • Ultrafiltration is a membrane filtration in which hydrostatic pressure is applied which causes movement of solution across the semi-permeable membrane.
  • Water and low molecular weight solute pass while other high molecular weight of molecules trapped in membrane.
  • The protein molecules are adsorbed in the membrane surface.
• Cell lysis: (For intracellular protein)
  • The intracellular proteins are liberated by cell lysis.
  • There are different methods for cell lysis.
  • They are:
  • Physical method:
  • Mechanical method: Bead mill, Homogenizer, Microfluidizer, Sonicator, French press/ X-mesh
  • Non-mechanical method: Decompression, Osmotic shock, Thermolysis, Freeze thaw, Dessication, Cell bomb
  • Chemical method:
  • Chemical permeabilizer whents
  • Antibiotics
  • Detergents
  • Chartrops
  • Chelating agents
  • Hydroxides and hypochlorides
  • Enzymatic method:
  • Autolysis
  • Lytic enzyme
  • Phage mediated lysis
• After cell lysis, the cellular constituents are concentrated by ultrafiltration.
• Refolding:
  • The first step in the refolding is the dissolution of the inclusion bodies (obtained from concentration) in a strong chaotropic solution of 6M urea, 2M thiourea.
  • Chaotropic agents are denaturating agent.
  • Chaotropic agents disrupt the intramolecular force between water molecules and allows protein and other macromolecule to dissolve easily.
  • The denaturated protein is then allowed to renature by removing the chaotropic agent by dilution, dialysis or by chromatographic separation.

V. Final purification:

• Chromatography is the usual method for obtaining pure protein.
• There are different types of chromatographic methods such as:
• Ion-exchange chromatography:
  • In case of ion exchange chromatography, cation or anion is attached to resin beads, depending upon the electric property of proteins.
  • If the desired protein is -vely charged then +ve charged resin beads are used.
  • The resin beads is packed in the column.
  • When the sample is poured in the column, the -vely charged protein (desired protein) stick on the beads while other undesired +vely charged protein eluted first.
  • The desired protein (-ve) is obtained as elute by changing the pH of the wash buffer or by washing with high salt solution.
• Hydrophobic chromatography:
  • This chromatography was developed to purify proteins by exploiting their surface hydrophobicity.
  • Groups of hydrophobic residues are scattered over the surface of proteins in such a way that it gives characteristic property to each protein.
  • The hydrophobic groups are covered by ordered layer of water in aqueous solution.
  • When salt is added then hydrophobic groups are exposed and interact with each other.
  • In hydrophobic interaction chromatography, the column is packed with hydrophobic beads (-phenyl, -acetyl group).
  • When the sample is poured, the hydrophobic protein interacts with hydrophobic matrix.
  • The salting-out compound such as ammonium sulfate is used from high to low concentration in the column so the protein with low hydrophobicity elute first.
  • The non-ionic detergents such as tween-20, triton-x-100 etc. are used to elute the protein.
• Affinity chromatography:
  • In affinity chromatography, a compound having specific affinity to desired protein is attached to the resin. For. e.g. Antibody against desired protein is coated on resin.
  • The resin is then packed into a column. When mixture of protein is poured, only those proteins having specific affinity with resin (Ab coated) stick on the column.
  • All the other protein gets eluted.
  • Only the undesired protein gets eluted.
  • The protein of interest stuck on the column can be eluted by changing the ionic strength of the solution so that the desired protein no longer binds to resin and get eluted.
  • This can also be achieved by adding special compound on elution solution which change the equation state and elute the protein.
• Size-exclusion chromatography:
  • This process is also known as gel filtration.
  • The method used to separate proteins on the basis of their size or molecular weight.
  • The porous matrix is packed in the column.
  • The porous matrix retards the rate of elution of proteins.
  • The protein with higher molecular weight elutes first since small protein passes through pores and elute last.

Protein characterization:

• The methods of protein characterization are:

i. Electrophoresis:

• It is the process of separation of charged particles under the influence of electric field.
• SDS-PAGE is the most widely used method for analysis of protein in the mixture.
• It is useful for monitoring the protein purification.
• SDS-PAGE separates proteins on the basis of molecular weight.
• At first polyacrylamide gel is made and a well is made.
• The protein sample is mixed with beta-mercaptoethane and sodium dodecyl sulphate (SDS) and boiled for 5 minutes.
• During boiling, proteins get denatured.
• Each SDS molecule binds to two amino-acids molecules of denatured protein.
• SDS molecule is highly -vely charged so the protein binds with SDS become -vely charged.
• When electrophoresis is done, the protein moves towards anode (+ve charge).
• The small size protein migrate faster and large size moves slower forming different band.
• The band can be visualized by staining with Coomassie brilliant blue (CBB).

ii. Peptide sequencing:

• This method is developed by Pehr Edman so it is also known as Edman degradation.
• The polypeptide is reacted with phenylisothiocyanate under mild alkaline condition.
• The amino terminal of peptide is converted to phenylthiocarbomyl (PTC).
• The phenylthiocarbomyl (PTC) derivatives is washed thoroughly with organic solvent (e.g. benzene) and dried.
• The dried PTC is treated with anhydrous acid (e.g. heptafluorobutyric acid).
• This results in cleavage of PTC-polypeptide near PTC substitution releasing N-terminal aminoacid as thioazoline derivatives.
• The thioazoline derivative is stable. So, it is converted to thiohydantoin derivative containing aminoacid is identified by high performance liquid chromatography (HPLC).
• If the aminoacid is alanine then the first aminoacid is the polypeptide along N-terminal is alanine.
• The Edman degradation process is repeated for sequencing other amino-acids.

iii. Tryptic mapping:

• Edman degradation method for determining amino-acid sequence from N-terminal require free amino-group at N-terminal of protein.
• However, 50-70% of proteins have N-terminal blocked by Formyl, acetyl or acryl group during post translational modification.
• For such protein, sequencing is not possible so, the protein have been cleaved by endopeptidases to produce peptide which is then sequenced.
• Trypsin enzyme is an example of endopeptidases which cleaves C-terminal of arginine and lysine.
• Similarly, other endopeptidases have their own restricted cleavage site.
• The generated short fragment is sequenced by Edman method followed by HPLC to identify the aminoacids.

iv. Analytical ultracentrifugation:

• This method measures variety of properties of protein sample including molecular weight, interaction with other molecules and sample homogeneity.

v. Spectroscopy:

• It is used in analysis of wide range of sample.
• The metal containing protein (co-factors) can be analysed by spectroscopy.
• The different co-factor gives different electromagnetic spectrum.

vi. Biosensors:

• It is a device used for the detection of particular protein in cell.
• The device is coated with specific Ab against the desired protein.
• When sample is added, the particular protein binds the Ab producing signal on device.

vii. Mass spectroscopy:

• Mass spectroscopy is an analytical technique that provide information about molecular structure of organic and inorganic compound.
• The mass of particular protein can be determined by mass spectroscopy.
• It can detect post translational modification or any variation in structure or protein.

Protein purification and characterization