Protein Production and Purification
Enzymes, antibodies and other proteins can be purified from natural sources such as cells, tissues or plants, or produced recombinantly in bacteria, yeast and mammalian cell culture or in the baculovirus insect cell system (See Molecular Biology and Cell Biology). Fermentation lot sizes vary up to 1gm of specific recombinant protein.
The desired protein can be purified to homogeneity (see below). Protein identity can be determined by a biological or enzyme activity, immunoassay, and amino acid sequence. Quality control can use a variety of biochemical assays.
Proteins are purified from cell supernatants, cell extracts, tissue homogenates, or E.coli cytoplasm or inclusion bodies by various preparative precipitation, centrifugal, electrophoretic, filtration and chromatographic steps. Salt precipitation, typically with ammonium sulfate, is a convenient and gentle first step and reduces the large volume of the starting material. Subsequent steps may use centrifugation, affinity purification on an antibody or substrate column or other affinity supports and a combination of size, charge, and hydrophobic chromatography. Final steps may include reverse phase HPLC. The desired protein is identified by a bioassay or enzyme activity, immunoassay and amino acid sequencing, and quality controlled by a variety of assays (See below and Immunology). Endotoxin is measured and removed if necessary.
Any given antibody (protein) can be separated from other proteins based on its unique physical and chemical properties. Proteins in solution form hydrogen bonds with water through their charged and polar side chain groups. If the protein solvent interaction is prevented, proteins can interact with each other and form aggregates that precipitate out of solution. As the concentration of salt is increased in a solution, the amount of water available to interact with protein is reduced, leading to interaction between hydrophobic groups on different proteins and the formation of a precipitate. Various factors, including the molecular weight of the protein, pH of the solution and temperature can affect the concentration at which a particular protein will precipitate out of solution. Ammonium sulfate is the salt most commonly used to precipitate proteins from solution. Organic, water miscible solvents (ethanol, methanol, acetone) can also be used to differentially precipitate out proteins.
A variety of chromatographic methods are performed. These may include size exclusion chromatography for native protein size, ion exchange, hydrophobic, reverse-phase based upon solubility separation, dye-binding and other chromatographic methods to characterize proteins.
High Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques. It utilizes a liquid mobile phase to separate the components of a mixture by forcing the components (analytes) dissolved in solvent to flow through a chromatographic column under high pressure. The mixture is resolved into its components based on the degree of interaction between the solute components and the stationary phase, defined as the immobile packing material in the column. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. The various components in the mixture pass through the column at different rates due to differences in their distribution between the mobile liquid phase and the stationary phase. Each protein has a characteristic peak under given chromatographic conditions and should have a reasonable retention time and be well separated from other extraneous peaks. Typically the protein is detected using ultraviolet spectrophotometry.
Ion Exchange Chromatography
Ion exchange chromatography separates proteins or peptides based on charge characteristics. The net surface charge of a protein or peptide determines its adsorption to oppositely charged groups immobilized on the ion-exchange medium. Proteins are multivalent anions or cations, and the charge of a protein depends on the pH of the environment. When the pH is greater than the isoelectric point (pI) of the protein (number of positive charges equals the number of negative charges), the protein will have a net negative charge and will bind to an anion exchanger. When the pH is less than the isoelectric point, the protein will have a net positive charge and will bind to a cation exchanger. Once the sample is bound to the medium, unbound components are washed away and bound samples selectively eluted and collected.
Affinity chromatography is frequently used with antibodies and recombinant proteins containing a “tag” which bind to specific ligands. Affinity columns can be made by crosslinking the ligand to the support gel, such as Sepharose, using a covalent attachment to a spacer arm. Typically, good separations of the protein from contaminants are achieved so that only one or two subsequent methods will resolve the protein to homogeneity.
Stable Formulation Development
There are a variety of strategies to stabilize proteins and enzymes. Storage of the proteins is investigated including lyophilization, flash freezing, normal freezing methods, and storage below 0 C with glycerol or DMSO like compounds. If kept at 2-8 C, the types of antibacterial reagents that can be used is determined. If kept at 2-8 C without antibacterial reagents, the method of generating a sterile protein solution without adsorption loses onto a filter is identified. The addition of stabilizing reagents is methodically studied so that bioactivity and physical conformation is not affected.
Protein concentration is determined by absorbance at 280nm (aromatic amino acids) or 205nm (peptide bond), by the Bradford assay or by amino acid analysis.
Purity is assessed by SDS-PAGE and/or nondenaturing electrophoresis for molecular weight and disulfide polymerization. Isoelectric focusing can be performed for identifying the isoelectric point (pI) of a protein. Other methods can be developed upon request.
Electrophoresis describes the migration of charged particles under the influence of an electric field. It can be used to evaluate protein purity and provide an estimation of characteristics such as isoelectric point, charge, and subunit composition. Gel electrophoresis is the technique in which molecules are forced across a span of gel by an electrical current. Proteins, peptides, amino acids, nucleotides etc. contain groupsthat can ionize and at any given pH, exist in solution as electrically charged species either as cations (+) or anions (-). Separation of large (macro) molecules depends upon two forces: charge and mass. During electrophoresis, the rate of migration in the electric field depends on the strength of the field, relative hydrophobicity of the samples, size and shape of the molecules, and on the ionic strength and temperature of the buffer in which the molecules are moving. The most common one dimensional gel methods use sodium dodecyl sulfate (SDS), which binds to proteins in a uniform amount per microgram of protein. This results in a uniform charge density per unit mass, providing a separation based on the mass of the polypeptide chain.
Proteins can be separated for further purification by isoelectric focusing (IEF) in slab gels in which the movement of proteins through pores in a polyacrylamide gel matrix is controlled by a pH gradient created by soluble ampholytes.
Binding Assays, Kd
A protein is characterized by binding to its receptor or ligand. The protein is bound to a solid support (ELISA plate, beads, column), incubated with labeled ligand, and the amount of bound ligand is determined. Equilibrium dialysis can be performed for precise Kd affinity values when applicable. Alternatively, the receptor may be on a cell surface. The affinity of the interaction, measured by the dissociation constant, Kd, is determined. These analyses may involve a UV spectophotometric, colorimetric, fluorescence or radioactivity method of detection.
Amino Acid Analysis and Sequence
The N-terminal amino acid sequence of a purified protein is determined to confirm its identity, the integrity of the N-terminus, and to verify that the preparation is homogeneously pure. Total amino acid analysis is performed to confirm the identity and full length of the protein and its absorbance at 280 nm.
Glycoproteins are specifically stained after SDS-PAGE fractionation. Deglycosylation can be performed as well as further analysis.
The limit of solubility and adjustment of salt, pH and formulation to increase solubility are performed. Solubility in a variety of detergents are investigated to retain protein or enzyme activity and conformation when developing isolation methods from cell membranes.
Enzyme Assays And Immunochemistry
High Throughput Screening Assays (HTS)
High throughput screening assays enable the screening of thousands of compounds for their activity or intervention in biological processes. Development of high throughput assays initially must demonstrate in a non-high throughput format that they are robust and reproducible with a high signal-to-noise ratio. Simple readouts of fluorescence, luminescence, colorimetric or radioactivity are used for molecular and biochemical reactions and are easily miniaturized. We can develop high throughput screening assays for your detection system and either transfer them or perform the screening for you.
Marin Biologic is experienced in many types of assays, including those employing proteases, dehydrogenases, oxidases, phosphorylase, nucleic acid polymerase and modifying enzymes, and glycolytic enzyme assays. Colorimetric, fluorescent and radiometric formats are used. Substrate specificity, ED50. ID50, substrate and inhibitor kinetics, temperature dependence and sensitivity are factors in selecting an assay. Assays for glucose, glutamine, lactic acid, endotoxin, etc. are also performed.
Enzyme and Protein Stability
Enzymes and proteins are tested for stability to temperature, pH, chemical denaturation, and proteolytic degradation. Methods for stabilizing enzymes and proteins during storage are developed.
ImmunoAssays (See Immunology)
The following immunoassays are discussed in the immunology section: ELISA, Enzyme-Linked ImmunoSorbent Assay
- Western Blot, Dot Blot
Other Biochemistry Methods
Data Base Characterization
Protein, peptide or coding DNA sequences are searched against databases to determine uniqueness, related families of proteins, and to suggest characteristic properties of a protein.
The Limulus Amebocyte Lymph (LAL) assay is used to quantitate pyrogen (endotoxin). Methods are used to remove endotoxin for biological assays.