Soluble Recombinant Proteins

Recombinant proteins that are not expressed in inclusion bodies either will be soluble inside the cell or, if using an excretion vector, will be extracellular (or, if E. coli is the host, possibly periplasmic). They can be purified by conventional means. In some systems, expression is so good that the desired product is the major protein present and its purification is relatively simple. In systems where the expression level is low, the purification process can be tedious, though easier, it is hoped, than isolation from the natural source. It should be remembered that a procedure developed for isolating a protein from natural sources may not work successfully with the recombinant product, because the nature of the other proteins present influences many fractionation procedures. Because of the difficulties often experienced in purifying recombinant proteins, a variety of vector systems have been developed in which the expressed prod-uct is a fusion protein containing an N-terminal polypeptide that simplifies purification. Such "tags" can be subsequently removed using a specific protease. A further advantage is that the expression level is dictated mainly by the transcription and translation signals for the fusion portion of the protein, which are optimized. Tags used include proteins and polypeptides for which there is a specific anti- body, binding proteins that will interact with columns containing a specific ligand, polyhistidine tags with affinity to immobilized metal columns, sequences that result in biotinylation by the host and enable purification on an avidin column, and sequences that confer insolubility under specified conditions. Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability- for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70°C for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). The host bacteria used for production of recombinant proteins are usually Escherichia coli, or Bacillus subtilis; they may express proteins at 1% to over 50% of the cellular protein, depending on such variables as the source, promoter structure, and vector type. Generally the proteins are expressed intracellularly, but leader sequences for excretion may be included. In the latter case, the protein is generally excreted into the periplasmic space, which limits the amount that can be produced. Excretion from gram-positive species such as B. subtilis sends the product into the culture medium, with little feedback limitation on total expression level.

Source from: http://protein-production.biologicscorp.com/strategies-for-native-protein-and-recombinant-protein-purification.html

Protein in diet

Proteins are the building blocks of life. The body needs protein to repair and maintain itself. The basic structure of protein is a chain of amino acids. Every cell in the human body contains protein. It is a major part of the skin, muscles, organs, and glands. Protein is also found in all body fluids, except bile and urine. You need protein in your diet to help your body repair cells and make new ones. Protein is also important for growth and development during childhood, adolescence, and pregnancy.

When proteins are digested, amino acids are left. The human body needs a number of amino acids to break down food. Amino acids need to be eaten in large enough amounts for optimal health. Amino acids are found in animal sources such as meats, milk, fish, and eggs, as well as in plant sources such as soy, beans, legumes, nut butters, and some grains (such as wheat germ). You do not need to eat animal products to get all the protein you need in your diet. Essential amino acids cannot be made by the body, and must be supplied by food. They do not need to be eaten at one meal. The balance over the whole day is more important.

Source from: http://www.nlm.nih.gov/medlineplus/ency/article/002467.htm

Protein Purification Methods Based on Bioproperties (Affinity)

Protein Purification Methods Based on Bioproperties (Affinity) : A powerful method for separating the desired protein from others is to use a biospecific method in which the particular biological property of the protein is exploited. The affinity approach is limited to proteins that have a specific binding property, except that proteins are theoretically able to be purified by immunoaffinity chromatography, which is the most specific of all affinity techniques. Most proteins of interest do have a specific ligand: enzymes have substrates and cofactors, and hormone-binding proteins and receptor molecules are designed to bind specifically and tightly to particular hormones and other factors. Immobilization of the ligand to which the protein binds (or of antibody to the protein) enables selective adsorption of the desired protein in the technique known as affinity chromatography. There are also nonchromatographic modes of exploiting biospecific interactions.

 
Source: protein purification blog

Overview of Protein Purification and Characterization

AIMS AND OBJECTIVES
Protein purification has an over 200-year history: the first attempts at isolating substances from plants having similar properties to “egg albumen,” or egg white, were reported in 1789 by Fourcroy. Many proteins from plants were purified in the nineteenth century, though most would not be considered pure by modern standards. A century later, ovalbumin was the first crystalline protein obtained (by Hofmeister in 1889). The year 1989 may not go down in history as a milestone in protein chemistry, but since then there has been a resurgence of interest in proteins after more than a decade of gene excitement.

The aims of protein purification, up until the 1940s, were simply academic. To then, even the basic facts of protein structure were not fully appreciated, and pure proteins were needed just to study structure and test the rival theories of the pre-DNA days. During the Second World War, an acute need for blood proteins led to development of the Cohn fractionation procedure for purification of albumin and other proteins from serum (Cohn et al., 1946). This was the inception of large-scale protein purifications for commercial purposes; Cohn fractionation continues to be used to this day.
The nature of the proteins studied has also changed substantially. Whereas enzymes were once the most favored subjects, they have now been superceded by nonenzymatic proteins such as growth factors, hormone receptors, viral antigens, and membrane transporters. Many of these occur in minute amounts in the natural source, and their purification can be a major task. Heroic efforts in the past have used kilogram quantities of rather unpleasant starting materials, such as human organs, and ended up with a few micrograms of pure product. It is now more usual, however, to take the genetic approach: clone the gene before the protein has been isolated or even properly identified, and then express it in a suitable host cell culture or organism. The expression level may be orders of magnitude higher than in the original source, which will make purification a relatively simple task. It can be useful to know beforehand some physical properties of the protein, to facilitate the development of a suitable purification protocol from the recombinant source. On the other hand, there are now several ways of preparing fusion proteins, which can be purified by affinity techniques without any knowledge of the properties of the target protein. Moreover, there are ways of modifying the expressed product to simplify purification further.

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