BL/CH401 Biochemistry
Recombinant proteins are made from cloned DNA sequences which usually encode an enzyme or protein with known function. Special vectors (a vector is simply a tool for manipulating DNA and can be viewed as a versatile "carrying vehicle") are used for production of the proteins from specific DNA sequences cloned into them. Since purification of a protein can be a complex and time-consuming process, it is often possible to add a DNA sequence to the cloned protein's code which will append a small peptide or even a small protein for facilitating the purification of the recombinant protein after it is expressed.
Since the N-terminal region of a protein or enzyme is often not too important to its functionality, a peptide sequence can be added as an extension at the N-terminal. This is easy to do by recombinant DNA methods, but actually comes as a built-in add-on to many commercially available vectors. So a researcher can select a purification system he or she would like to use and simply buy the vector which has what is needed to do the job. These special vectors have a number of features needed for a producing a high level of the target protein in a specific organism like E. coli. In addition, the purification tool is placed in the vector so that it is added to the N-terminal of the target protein but separated by a amino acid sequence designed to be a point of attack for a specific protease. Thus, after the recombinant protein is expressed and extracted from E. coli, the N-terminal extension can be used to purify the protein and subsequently removed from the N-terminus to generate a nearly natural N-terminus sequence on the final product. The latter step of removing the unnatural portion of the sequence may be very important, for example if the target protein is to be used as a drug.
Metal Chelate Affinity Chromatography: More than 20 years ago it was discovered that many natural proteins have metal binding sites which can be used for purification. The concept of this type of purification tool is rather simple. A gel bead is covalently modified so that it displays a chelator group for binding a heavy metal ion like Ni2+ or Zn2+. The design of the chelating group on the gel bead is such that it provides only half of the ligands needed to hold the metal ion. So when the protein with a metal binding site finds the heavy metal, the protein will bind by providing ligands from its metal binding site to attach to the metal ion displayed on the chelator arm of the gel bead. This is very similar to affinity chromatography and can be viewed as a group selective tool for purifying the metal-binding class of proteins.
Illustration of Metal Chelate Affinity Chromatography Procedure for Purifying a Metal Ion Binding Protein
His-Tag for Purification of Recombinant Proteins: It has been shown that an amino acid sequence consisting of 6 or more His residues in a row will also act as a metal binding site for a recombinant protein. So a hexa-His sequence is called a His-Tag. A His-Tag sequence can be placed on the N-terminal of a target protein by using vectors from various commercial molecular biology companies. As illustrated below, the His-Tag is often followed by a cleavage site for a specific protease - for example LeuValArgGlySer peptide sequence is recognized and cleaved by the protease known as thrombin:
MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer....recombinant protein sequence
So the His-Tag recombinant protein can be purified by Metal Chelate Affinity Chromatography as illustrated above. Usually nickel ions are used as the heavy metal ion and the His-Tag protein is eluted from the metal-chelate column with His or imidazole. Then the purified His-Tag protein is treated with the specific protease to cleave off the His-Tag. Finally, the recombinant protein is freed of the His-Tag peptide by running it over the metal-chelate column again. This can be a very effective method to purify a recombinant protein for which there is no known easy way to purify using substrate-based affinity chromatography.
New England Biolabs (NEB) sells the Protein Fusion and Purification System for facilitating purification of recombinant proteins. The system uses maltose-binding protein (MBP) as a tool for purification. The gene for MBP is built into the pMAL vector which sold as part of the system. The other components necessary for doing the target protein purification are also included in the system kit. The system works as illustrated in the graphic shown below:
Graphic from NEB 96-97 catalog on P. 164, which is copyrighted by NEB.
The results of a purification of a recombinant protein (paramyosin) expressed with the MBP attached is shown below as analyzed by an SDS-PAGE gel. In part A, the crude extracts from uninduced cells and induced cells expressing the fusion protein of MBP and paramyosin are shown in lanes 1 & 2 respectively. In part B, the purified MBP-paramyosin fusion protein is shown after elution from the amylose column with maltose (Lane 1); then the two fused proteins are shown after separation by treatment with the protease called Factor Xa (Lane 2); and finally the pure paramyosin is shown after removal of the MBP by passing the mixture over another amylose column where the recombinant paramyosin simply passes through the column without binding (Lane 3).
Graphic from NEB 96/97 Catalog who owns copyright
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Bill Campbell © 1997; All Rights Reserved; wcampbell@mtu.edu