Protein Purification What is protein purification? Protein purification is the separation of a specific protein from contaminants in a manner that produces a useful end product. Why purify proteins? In a research environment, proteins must be purified in order to determine their structure and study their biochemical properties. In industrial settings, proteins are purified on a larger scale in order to be sold as products such as drugs, vaccines, diagnostic tools or food additives. Issues: purity, yield and cost

Separation methods are based on protein properties Separation methods are based on protein properties. Because of the differences in amino acid composition and sequence, and the possible presence of non-protein groups, each protein has different chemical characteristics that make it unique. These characteristics include size (molecular weight) Charge solubility hydrophobicity biological affinity Differences in these characteristics are the basis for separation methods such as Filtration salt precipitation chromatographic procedures

Protein could be included inside the cell or secreted out of the cell. Depending on which you will need to isolate the cells or the media. Generally achieved by centrifugation or possibly filtration. If the protein is within the cells the next step needed is lysis of cells.

Techniques used for the physical disruption of cells. Lysis Method Description Apparatus Mechanical Waring Blender Polytron Rotating blades grind and disperse cells and tissues Liquid Homogenization Homogenizer French Press Cell or tissue suspensions are sheared by forcing them through a narrow space Sonication Sonicator High frequency sound waves shear cells Freeze/Thaw Freezer or dry ice/ethanol Repeated cycles of freezing and thawing disrupt cells through ice crystal formation Manual grinding Mortar and pestle Grinding plant tissue, frozen in liquid nitrogen Lysis methods 1.Physical methods -Bead beater-use glass beads 0.1 -0.2 mm, -Homogenizer -Grinder (freeze sample –liquid nitrogen) -Mortar and pestle-use liquid nitrogen

-Lysozyme with or without EDTA to digest the peptidoglycan layer Lysis methods cont. 2. Chemical methods -chloroform or toluene-to solubilize the membrane, alkaline NaOH, detergent, extreme salt concentrations 3. Enzymatic methods -Lysozyme with or without EDTA to digest the peptidoglycan layer Next the protein needs to be separated either from the contents of the spent media or from the contents of the lysed cell. Detergents break the lipid barrier surrounding cells by solubilizing proteins and disrupting lipid:lipid, protein:protein and protein:lipid interactions. Detergents, like lipids, self associate and bind to hydrophobic surfaces. They are comprised of a polar hydrophilic head group and a nonpolar hydrophobic tail and are categorized by the nature of the head group as either ionic (cationic or anionic), nonionic or zwitterionic. Their behavior depends on the properties of the head group and tail.

A variety of methods are used to separate out the protein , including some of the following: 1. Filtration In Ultrafiltration, molecules such as proteins and nucleic acids are retained by the filter. These filters can only separate very large proteins from very small proteins; they are mainly used for concentrating proteins and for exchanging buffers. 2. Protein Precipitation This step is used at an early step on crude material. A protein precipitate will form when proteins are prevented from interacting with the surrounding water molecules e.g. "Salting Out": Salts such as ammonium sulfate., changing pH, heat denaturation

3. Ion exchange chromatography Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups) which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge.

A basic protein, which is positively charged, will bind to a support which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidics, basics and neutrals) can be easily separated by this technique. A very frequently used chromatographic technique for protein purification. Matrix example DEAE

3. ion-exchange chromatography allows molecules to be separated based upon their charge

4. Hydrophobic Interaction Chromatography Not all of the common amino acids found in proteins are charged molecules. some amino acids that contain hydrocarbon side-chains which are not charged cannot be purified by ion-exchange chromatography. These hydrophobic amino acids are usually buried away in the inside of the protein as it folds into it's biologically active conformation. Some distribution of these hydrophobic residues on the surface of the molecule.

These hydrophobic amino acids can bind on a support which contains immobilized hydrophobic groups. HIC supports work by a "clustering" effect; no covalent or ionic bonds are formed or shared when these molecules associate. Hydrophobic residues are alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, proline. High salt exposes the hydrophobic groups which then bind to the matrix. Elution is carried out by lowering the salt concentration or with organic solvents if necessary. Matrix examples: phenyl or octyl sepharose

4. Hydrophobic Interaction Chromatography

separates proteins based on size and shape. 5. Gel-Filtration Chromatography separates proteins based on size and shape. The support for gel-filtration chromatography are beads which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces which separate the beads faster than the smaller molecules, which may penetrate the pores. The matrix pore size determines the rate at which different proteins can diffuse, and some proteins will be completely excluded. The pore size chosen will Depend on the specific protein to be purified. The column is eluted with buffer and the proteins come out in order of the largest first. Matrix example: Sephadex

6. Affinity Chromatography It is the only technique which can potentially allow a one-step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. E.g. the use of an immobilized protein to capture it's receptor (the reverse would also work). Can be used for the purification of any protein, provided that a specific ligand is available. Ligand examples: a. Lectins such as wheat germ agglutinin or ConA which bind to the carbohydrate portion of proteins b. Protein A or G which binds the Fc regions of some immunoglobulins c. Metal chelate chromatography — Zn columns bind histidine tagged proteins. d. Dyes such as blue dextran