Historically, proteins have been isolated directly from organisms, tissues or fluids in a non-recombinant manner. The ability to clone and over-express genes for protein synthesis using genetic engineering methodology has changed this dramatically and nowadays the vast majority of protein isolation is carried out using recombinant methods with over-expression in bacterial or eukaryotic cells. Therefore, the manipulation of the gene of interest to engineer particular protein constructs is the first important step in the design of a protein purification procedure.

Some typical manipulations that are carried out to aid purification, improve folding or ensure secretion of recombinant proteins are discussed below.

Fusion Constructs to Aid Protein Purification

This approach requires the introduction of a genetic sequence that codes for a small peptide, or a well-characterised protein, to be placed in frame with the protein of interest, such that the protein is produced as a fusion protein. The peptide or protein (known as a ‘ tag’) can be placed at the N- or C-terminal ends and provides a means for the fusion protein to be selectively purified from the cell extract by affinity chromatography. A protease site is typically engineered between the tag and protein that is then cleaved to release the protein of interest from the fusion construct. Clearly, the amino-acid sequence of the peptide linkage between tag and protein has to be carefully designed to allow chemical or enzymatic cleavage of this sequence. There are more than 20 published protein and peptide tags and each has specific characteristics beneficial to a particular experiment; the following sections describe some of the most commonly used tags.

Glutathione- S -transferase (GST)

 The protein of interest is expressed as a fusion protein with the enzyme glutathione- S -transferase ( GST) that was originally cloned from the blood fluke Schistosoma japonicum. The cell extract is typically passed through a column packed with glutathione-linked agarose beads, whereupon the GST binds to the glutathione with high affinity. Wash buffer is applied to the beads and once the non-tagged protein is washed from the column, the GST-fusion protein is eluted by adding an excess of reduced glutathione (a tripeptide with a γ-peptide linkage, γ-Glu-Cys-Gly), displacing the bound GST moiety. A typical further step would be removal of the fusion protein. In the pGEX series of vectors, this is achieved using human thrombin, which cleaves a specific amino-acid sequence contained within the linker region ( Table 1). GST is typically fused via the N-terminal to the target gene and the fusion constructs often possess better solubility than the native proteins, albeit this effect is not as pronounced as with maltose-binding protein. However, GST does contain four cysteine residues that can become oxidised and cause aggregation, which can be a problem for expression of oligomeric proteins.

Table1. Summary of commonly used proteases in protein production techniques

Maltose-Binding Protein (MBP)

Maltose-binding protein has the desirable property of significantly enhancing the solubility of the target protein. MBP is itself a well-ordered protein and is believed to aid the folding of the target protein, if placed on the N-terminal side. As such, it is often employed when attempting to express ‘difficult’ proteins that fail to be expressed when attempted with a small tag such as poly-histidine or GST. One-step purification can be achieved using amylose resin (that binds MBP), although care has to be taken to avoid amylose degradation by galactases. This is achieved by supplementing the media with 2 mM glucose to the growth media, as this represses galactase expression.

Poly-Histidine Tag (His-tag)

The essential amino acid histidine contains an imidazole ring that provides a stable structure, enabling co-ordinated binding with metal ions in a pH-dependent manner. The His-tag typically contains six sequential histidine residues that confer binding to immobilised bivalent nickel or cobalt ions with a dissociation constant in the μM range. The captured protein is efficiently eluted with either free imidazole (typically at 50–300 mM) or at acidic pH. Low concentrations of imidazole (10–20 mM) may be included in the wash buffer to increase purity of the final eluate. An extension to this method is the introduction of two hexa-histidine tags in series; this increases the affinity to the capture media to allow more aggressive washing. Correspondingly, the concentration of free imidazole required to achieve elution needs to be increased, typically to 300 mM.

FLAG ®

This is a short hydrophilic amino acid sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) that may be attached to the N- or C-terminal end of the protein, and is designed for purification by immunoaffinity chromatography. A monoclonal antibody against the FLAG ® sequence is available on an immobilised  support for use in affinity chromatography. The cell extract, which includes the FLAG ® -labelled protein, is passed through the column where the antibody binds to the FLAG ® -labelled protein, allowing all other proteins to pass through. This is carried out in the presence of calcium ions, since the binding of the FLAG ® sequence to the monoclonal antibody is calcium- dependent. Once all unbound protein has been washed from the column, the FLAG ® linked protein is released by passing EDTA through the column, which chelates the calcium ions. If the FLAG ® sequence is used as an N-terminal fusion, it can conveniently be removed by the enzyme enterokinase, which recognises the poly-aspartate sequence and cleaves on the C-terminal side of the following lysine residue (see Table 1). In some cases, it can be advantageous to leave the tag attached, such as to enable convenient tracking of the protein in downstream experiments.

Dual Tags

Combinations of tags are sometimes used to allow tracing of protein using a convenient method, such as Western blotting, or improved protein production. A commonly used combination is the fusion of the hexa-histidine tag and maltose-binding protein (MBP) used in tandem, which brings together two advantageous properties. The His tag allows economical and rapid purification and the MBP increases the solubility of the protein. Frequently, a TEV protease site is engineered between the His–MBP fusion and the target protein to allow efficient removal of the tags with minimal cost.

AviTag ^TM (Acceptor Peptide)

This 15 amino-acid sequence was optimised by phage display and the sequence can be efficiently biotinylated in vitro using the Escherichia coli enzyme BirA, which conjugates D -(+)-biotin to the lysine residue indicated:

Biotinylation is also possible in transfected mammalian cells. The biotin group is recognised by streptavidin ; the complex of both proteins associates with a dissociation constant ( Kd ) of 4 × 10⁻¹⁴ . As such, this method can be used to efficiently purify in vivo tagged proteins that are natively folded and post-translationally modified. The biotinylated protein may also be useful for a number of applications such as binding assays, or detection of biomolecules.

Proteases Used in the Purification Process of Engineered Proteins

Some commonly used proteases for the removal of fusion peptides and proteins are summarised in Table 1. A very popular protease is the tobacco etch virus (TEV) endopeptidase, which is patent-free and economically produced in laboratories in deference to commercially available enzyme systems such as Factor X, etc.

In order to overcome the relatively poor solubility of TEV protease, it is common to express it in bacterial culture as an MBP fusion construct with a TEV protease recognition site. During expression, the produced protein construct cleaves the MBP fusion protein, resulting in the target protease. As TEV protease is prone to autolysis, a single amino-acid change (S219V) is typically engineered and sufficient to remove this property; the mutant is also slightly more efficient than the wild-type enzyme.

Purification pipeline considerations also led to addition of a His-tag to the TEV protease constructs. This not only aids in convenient purification of the enzyme in the laboratory, but also provides an elegant means for the final clean-up after proteolysis of a His-tagged target protein. With immobilised metal ion chromatography, the cleaved poly-histidine tag and His-tagged TEV protease, as well as any non-cleaved His-fusion target, are retained on the affinity column.

Intein-Mediated Purification

 As discussed above, it is possible to create fusion proteins by engineering the DNA to contain the required sequence. However, it may be desirable to introduce post- translational modifications that cannot be achieved through molecular cloning or reliably achieved through chemical or enzymatic modification. In a process known as intein-mediated protein ligation, or expressed protein ligation , two peptides or proteins are covalently linked to create a fusion protein. In this method, a protein is produced with a thioester at the C-terminus and ligated to a peptide or protein containing an N-terminal cysteine. This methodology utilises the inducible self-cleavage activity of protein splicing elements (termed inteins ) to separate the target protein from the affinity tag (see Figure1 ); there is thus no requirement for a protease.

Fig1. Intein-mediated protein ligation enables the covalent linking of two proteins/peptides via an N-terminal cysteine residue on the conjugation partner.

An example application of this technique is the study of protein phosphorylation, where, for the purpose of binding studies, it is essential to know the stoichiometry of phosphorylation. Intein-mediated fusion allows the generation of a protein of interest and its fusion with a separately synthesised phosphorylated peptide (where the degree of phosphorylation is known). This is achieved by separating the incomplete intein-mediated ligation protein from the successfully ligated protein, for example by size-exclusion chromatography.

Secreted Proteins

 For cloned genes that are being expressed in microbial or eukaryotic cells, there are a number of advantages in manipulating the gene to ensure that the protein product is secreted from the cell:

• To facilitate purification : Clearly if the protein is secreted into the growth medium, there will be far fewer contaminating proteins present than if the cells had to be ruptured to release the protein, when all the other intracellular proteins would also be present.

 • Prevention of intracellular degradation of the cloned protein : Many cloned proteins are recognised as ‘foreign’ by the cell in which they are produced and are therefore degraded by intracellular proteases. Secretion of the protein into the culture medium should minimise this degradation.

• Reduction of the intracellular concentration of toxic proteins : Some cloned proteins are toxic to the cell in which they are produced and there is therefore a limit to the amount of protein the cell will produce before it dies. Protein secretion should prevent cell death and result in continued production of protein.

• To allow post-translational modification of proteins : Most post-translational modifications of proteins occur as part of the secretory pathway, and these modifications, for example glycosylation, are a necessary process in producing the final protein structure. Since prokaryotic cells do not glycosylate their proteins, this explains why many proteins have to be expressed in eukaryotic cells (e.g. yeast) rather than in bacteria. The entry of a protein into a secretory pathway and its ultimate destination is determined by a short amino-acid sequence (signal sequence) that is usually at the N-terminus of the protein. For proteins targeted to the membrane or outside the cell, the route is via the endoplamic reticulum and Golgi apparatus, the signal sequence being cleaved off by a protease prior to secretion. In some cases, secretion can be achieved by using the protein’s native signal sequence (example: human γ-interferon expression in Pichia pastoris ). In addition, there are a number of well-characterised yeast signal sequences (e.g. the α-factor signal sequence) that can be used to ensure secretion of proteins cloned into yeast.

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Introduction to Immunological methods

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