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PROTEIN PURIFICATION home > what do our scientists do > protein purification
Many molecular biologists and biochemists can use nanogram (10-9 gram) quantities of protein for their experiments. In order to carry out successful structure determinations, however, crystallographers require a million times more protein. HWI’s protein production and purification laboratory has been designed for the purpose of producing proteins on a 1-100 milligram (10-3 gram) scale and to achieve 99 percent purity for crystallization and subsequent structural studies.

Small-Scale Protein Expression: Protein expression is affected by many experimental variables such as temperature, cell density, rate of growth, cell type, chemical inductants, and induction lengths. The process is further complicated by the unique behavior and characteristics of each protein. For example, many proteins differ drastically in their solubility when expressed under different experimental conditions. New protein projects in the lab are generally subjected to small-scale (25 milliliter to 1 liter) experiments designed to identify conditions that produce the highest level of protein expression and the highest solubility.

Large-Scale Protein Expression: After optimal conditions for expression have been identified, the process is scaled-up (i.e. the amount of protein produced is increased) using fermentation. Many critical cell-growth parameters can be closely monitored and controlled with fermentor-based systems. Temperature, oxygenation, and pH levels are recorded and controlled by computer to ensure maximum protein expression. In addition, fermentors can handle any cell line and can be reconfigured quickly for new runs. This technology can be used to produce proteins in which the natural sulfur-containing amino acid methionine is replaced by its selenium-containing analog, selenomethionine. The presence of selenium is useful later when the diffraction data are analyzed.

Purification: Rapid purification of expressed proteins is accomplished using high-pressure liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC). In order to handle the large volume of cell lysate (disrupted cells) from the fermentors, affinity chromatography using purification tags expressed with the protein is the first step. Affinity columns usually can handle large volumes and flow at high rates. Further steps of purification (ion exchange, hydrophobic and gel filtration) occur at an analytical level and are based on the individual characteristics of the proteins studied. The FPLC can be set up to direct the peaks from one column directly to another, thereby creating an automated purification system. Careful control of temperature, pressure, and flow rates are characteristic of these HPLC and FPLC systems and makes them ideal for high throughput purification.

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