Proteins are constantly being created by the cellular machinery of living organisms. This article will first summarize the process as it occurs in a natural organism, and then discuss how protein expression and purification occurs in a laboratory setting for the generation of recombinant proteins.
In bacteria there are two main types of DNA—genomic and plasmid. Plasmid DNA is unique to bacteria. Eukaryotic cells don't typically have plasmid DNA unless it was put there by transfection for experimental purposes. The most important goal when isolating nucleic acids is to obtain the highest purity genetic material possible. When isolating genomic DNA it is important to remove plasmid DNA and RNA from the sample. Similarly, sometimes an experiment calls for the isolation of plasmid DNA, and the selective removal of genomic DNA is necessary. Also, some commercial RNA isolation kits include gDNA eliminator spin columns to remove genomic DNA from the isolate.
The concept of an antigen and antibody pair is central to modern medicine and biotechnology. These proteins match like a lock and key, with equisite specificity. The interactions are non-covalent, but have equilibrium constants ranging from 105 to 1012 M-1 Antibodies and antigens are proteins: polypeptide chains of amino acids. The IgG antibody is composed of four polypeptide chains, two heavy and two light, organized into a ‘Y’ shape. The base of the Y is called the Fc region, while the two tips are known as the Fab region.
Traditional immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are able to measure the presence or absence of only one analyte per reaction. Multiplex immunoassays measure dozens of different analytes in a single reaction. This is particularly beneficial for precious samples, and when only a small volume is collected for analysis. The multiplex immunoassay also saves working time since multiple assays can be completed simultaneously.
Immunoprecipitation (IP) is a method used to purify target proteins from whole tissue or from cell culture. There are different types of IP: Single protein, Co-immunoprecipitation (co-ip), Chromatin immunoprecipitation(CHIP), RNA immunoprecipitation (RIP), and tagged protein immunoprecipitation.
Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogenous solution. It is column-based, which means that the solution is flowed through a column and eluted at the other end. The column is pre-functionalized with the capture antibody or antigen. The target protein is adsorbed onto the resin-bound capture protein and is retained in the column while the remaining solution is eluted. The fraction containing the target protein is later eluted and purified.
Size exclusion chromatography is used to separate molecules by size, molecular weight, and hydrodynamic volume. The technique can be used with proteins, polymers, and other macromolecules. It can also be used for buffer exchange or desalting a sample. The principle behind size exclusion chromatography is simple, but the technique only works when the correct resin-bound column is matched to the experimental goal.
Enzymes are used in the food, agricultural, cosmetic, and pharmaceutical industries to control and speed up reactions in order to quickly and accurately obtain a valuable final product. Enzymes are crucial to making cheese, brewing beer, baking bread, extracting fruit juice, tanning leather, and much more. Enzymes are also increasingly being used in the production of biofuels and biopolymers. The enzymes can be harvested from microbial sources or can be made synthetically. Yeast and E. coli are commonly engineered to overexpress an enzyme of interest. This type of enzyme engineering is a powerful way to obtain large amounts of enzyme for biocatalysis in order to replace traditional chemical processes.