Add Xphos To Detect Location Of Secondary Antibody

Visible blue band indicates location of the protein

Sample molecule

Sample molecule

Matrix

Ionized sample molecule

Charged grid

FIGURE 26.07 MALDI/TOF Mass Spectrometer

Matrix

Ionized sample molecule

Charged grid

FIGURE 26.07 MALDI/TOF Mass Spectrometer

Mass spectrometry can be used to determine the molecular weight of proteins. The proteins are crystallized in a solid matrix and exposed to a laser, which releases ions from the proteins. These travel along a vacuum tube, passing through a charged grid, which helps separate the ions by size and charge. The time it takes for ions to reach the detector is proportional to the square root of their mass to charge ratio (m/z). The molecular weight of the protein can be determined from this data.

Solution of sample molecules

Droplet

Charged grid

FIGURE 26.08 Electrospray Ionization Mass Spectrometer

Solution of sample molecules

Droplet

Charged grid

FIGURE 26.08 Electrospray Ionization Mass Spectrometer

ESI mass spectrometry uses a liquid sample of the protein held in a capillary tube. After exposure to a strong electrostatic field, small droplets are released from the end of the capillary tube. A flow of heated gas within the drift zone helps evaporate the solvent and release small charged ions. The charged ions vary in size and charge and the pattern of ions produced is unique to each protein. The ions are further separated by size using a charged grid to either impede or help the flow toward the detector.

ions, so allowing more detailed analysis of molecules. This is known as tandem mass spectrometry (MS/MS). It allows two parent ions with the same mass (e.g. two peptides with the same amino acid composition but different sequence) to be distinguished.

Protein Tagging Systems

Tagging of proteins with another easily recognized molecule allows many different proteins to be processed and purified by the same procedures. Protein tagging is usually done genetically, that is, the DNA encoding the protein is engineered to add an extra segment that codes for the tag. The gene must therefore first be cloned and carried on a suitable vector. The hybrid gene is then expressed in a suitable host organism, such as Escherichia coli or cultured mammalian cells, and the protein is purified by a method that binds to and isolates the tag sequence. Since the protein of interest is synthesized attached to the tag, it is purified along with the tag molecule. In many instances, the tag portion may then be removed. Alternatively, a protein array can by constructed by attaching the tagged protein to a chip via the tag (see below).

The first protein tag to be widely used was the polyhistidine tag (His tag), consisting of six tandem histidine residues. This may be added to the target protein at either the amino or carboxy terminus. The His tag binds very tightly to nickel ions; therefore, His tagged proteins are purified on a column to which Ni2+ ions are attached by a metal chelator (metal-chelate column chromatography) (Fig. 26.09).

Other short tags in wide use are the FLAG® and "Strep" tags. The FLAG tag is a short peptide (AspTyrLysAspAspAspAspLys) that is bound by a specific antibody. Anti-FLAG® antibody is available commercially (Immunex Corporation) and may be attached to a suitable resin for use in column purification. The "Strep" tag is a 10 amino acid peptide that mimics the 3-D structure of biotin. It is bound by the biotin-binding proteins avidin or streptavidin and was originally selected from a random oligopep-tide library for its ability to bind to streptavidin.

Full-Length Proteins Used as Fusion Tags

Longer tags, consisting of entire proteins, are sometimes used to tag a specific protein. The tag protein coding sequence is normally placed 5' to the gene of interest in order to ensure good translation initiation. Three of the most popular are protein A from Staphylococcus, glutathione-S-transferase (GST) from Schistosoma japonicum, and maltose-binding protein (MBP) from E. coli.These three proteins generally give fusion protein products that are stable, soluble and behave well during purification.

The fusion proteins are purified on columns containing material that specifically bind the tag protein. A specific commercially available antibody binds protein A. After purification on an antibody column, the fusion protein can be eluted at pH 3. Glu-tathione-S-transferase binds to glutathione, a tripeptide. GST fusions are bound by glu-tathione-agarose columns and eluted with free glutathione. Maltose-binding protein avidin Protein from egg-white that binds biotin very tightly

FLAG® tag A short peptide tag (AspTyrLysAspAspAspAspLys) that is bound by a specific anti-FLAG® antibody that may be attached to a resin for use in column purification of proteins glutathione-S-transferase (GST) Enzyme that binds to the tripeptide, glutathione. GST is often used in making fusion proteins His tag Six tandem histidine residues that are fused to proteins so allowing purification by binding to nickel ions that are attached to a solid support. Also known as polyhistidine tag maltose-binding protein (MBP) Protein of E. coli that binds maltose during transport. MBP is often used in making fusion proteins polyhistidine tag (His tag) Six tandem histidine residues that are fused to proteins so allowing purification by binding to nickel ions that are attached to a solid support protein A Antibody binding protein from Staphylococcus that is often used in making fusion proteins streptavidin Protein from Streptococcus that binds biotin very tightly tandem mass spectrometry (MS/MS) Two successive rounds of mass spectrometry in which a parent ion is first isolated and then fragmented into daughter ions for more detailed analysis

A convenient way to identify and purify proteins is to tag them using a genetic approach.

The His tag allows proteins to be purified by binding to a resin containing nickel ions.

Proteins may be fused to a second protein chosen for its convenient properties.

Full-Length Proteins Used as Fusion Tags 727

FIGURE 26.09 Nickel Purification of His6 Tagged Protein

To isolate a pure sample of one specific protein, the gene for the protein is linked to a tag sequence. In this example, the tag sequence encodes six histidines in a row. This engineered gene is then expressed in either bacteria or mammalian cells. The cells are harvested and lysed to release all the proteins. To isolate the tagged protein, the mixture of proteins is added to a nickel column. The nickel-coated beads bind the histidine tag, allowing all the other proteins to pass through the column. Next a solution containing histidine or imidazole is added to the column. The histidine or imidazole binds to the nickel-coated beads thus releasing the histidine tagged protein.

Nickel column

Proteins poured through column

His tagged protens bind

Other proteins pass through

Elute WITH HISTidiNE OR IMIDAZOLE

His tagged protein is ^ displaced

His tagged protein is ^ displaced

HHHHHH«

HHHHHH«

FIGURE 26.10 Protein Isolation using Fusions to Maltose-Binding Protein

In order to isolate one specific protein from a mixture, the gene for maltose-binding protein can be genetically fused to the protein of interest. The fusion gene can then be expressed in an organism such as E. coli. The bacteria are lysed, and the mixture of proteins (top of figure) is isolated. The protein of interest has three new regions attached to it: the entire maltose binding protein, a cleavage site for Factor Xa, and a small spacer region. The protein mixture is added to an amylose column, where only the fusion protein binds via the maltose binding domain. Adding free maltose elutes the fusion protein. The eluted protein is treated with factor Xa, which cleaves the maltose binding protein section from the protein of interest.

binds maltose and the maltose polymer, amylose. An amylose column purifies proteins with an MBP tag. The bound fusion protein is then eluted with maltose, which preferentially binds to the amylose and releases the tagged protein (Fig. 26.10).

The pMAL vectors (New England Biolabs Inc., MA) are one example of a protein tagging system (Fig. 26.11).These vectors carry the malE gene of E. coli, encoding MBP. A spacer sequence encoding 10 Asn residues lies between malE gene and the polylinker. This insulates the maltose binding protein from the protein of interest. Adjoining this is the recognition site [Ile Glu (or Asp) Gly Arg] for a highly specific protease, factor Xa of the blood clotting system. After purification, the fusion protein is treated with a factor Xa and the two proteins are separated. Factor Xa itself must then be removed by binding it to benzamidine-agarose.The protein fusion is transcribed from the strong tac promoter and is translated using the initiation signals of malE. Variants exist with or without the malE signal sequence thus allowing for either secretion or cytoplasmic expression.

Sometimes, fusion proteins (or for that matter unmodified proteins) are degraded by host-cell proteases during purification. Protease inhibitors may be included, or in the case of a bacterial host such as Escherichia coli, protease-deficient mutants may be

MalE fusion protein

MalE fusion protein

Mixture of proteins

MalE FUSIon bINDS AmylosE

Mixture of proteins

MalE FUSIon bINDS AmylosE

Maltose

Other proteins pass through

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