Gene Expression And Protein Purification

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DNA is the genetic material of most of the organism. Gene is a segment of DNA responsible for the production of RNA and finally peptide. Gene expression is the process by which information from a gene is used in the synthesis of a functional gene products which are often proteins needed for the life. This article discuses and summarizes important work in the literature regarding the gene expression and protein purification. Much stress has been given to gene expression in bacteria, yeast, insects and mammals. The author also directed his studies towards protein purification. The study was mainly focused on the role of His-tag, GST-tag, MBP-tag, IMPACT, TAP-Tagging towards protein purification. Other strategies during protein purification were also highlighted. The article will be helpful for those researchers who are going to start their career in gene expression and protein purification.

Key words: Gene, Gene expression, Protein purification, Protein purification tag.


Each and every organism possesses the information required to construct and maintain a living copy of it self. All the genetic information is maintained within nucleic acid. The ability to direct genetic changes at the molecular level has resulted in a revolution in biology. The discovery of genetic code many years ago suggested that gene isolation and transfer into living organisms would become major tools for biologists. Protein engineering can be thought of as the deliberate modification of the sequence of protein through the alteration of the DNA sequence encoding it so to impart the protein with a new or novel function. Different types of proteins can be produced by gene expression using recombinant DNA technology.

A gene is described as a discrete unit of genetic information that is required for the production of a polypeptide. It includes the coding sequence, the promoter, terminator and introns. The synthesis and purification of protein from cloned gene is one of the most important aspects of genetic manipulation, particularly where valuable therapeutic proteins are concerned. In protein production there are two aspects which require optimization that is the biology of the system and the production process itself. For efficient expression of cloned DNA, the gene must be inserted into a vector that has a suitable promoter, and which can be introduced into an appropriate host. The vectors used for cloning a gene into it are listed in the Table 1.

A gene can be expressed in a number of expression systems. The expression level of a gene largely depends upon how efficiently it is transcribed. This article discusses and summarizes important work in literature regarding the systems which are appropriate for gene expression. Moreover it also highlighted the modern ways of protein purification after the expression of gene. This article will be a boon for the readers who are starting their career in the field of gene expression and protein purification. Prior to this work the author has reported about the vectors used in gene manipulation (Khan, 2009a). Moreover he also reported about gene transfer technologies in plants (Khan, 2009b) and also gene transfer technologies leading to transgenic animals(Khan, 2009c). In this article the author highlighted the expression of gene in bacteria, yeast, insect and mammals. Various strategies regarding protein purification were also discussed. The systems in which genes can be expressed to get protein are as follows.


In order to obtain a desirable level of expression, the cloned gene must be transcribed and translated efficiently. The choice of expression system plays an important role for the production and modification of protein to retain its biological activities. To maximize protein expression it is vital that an inducible expression system be established so to enable to grow large number of host cells before to initiate the expression of target protein. Various systems like gene expression in bacteria, insect, yeast and mammalian cell lines have been discussed.

Gene expression in bacteria

E. coli is one of the most widely used hosts for the production of heterologous proteins. Thegenetics of E.coli are far better characterized than those of any other microorganism. Recent development in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.

The advantage of this system is the ability to produce protein in large amount. The growth of E. coli is very fast in comparison to mammalian cells, giving the opportunity to purify, analyze and use the expressed protein in a much shorter time. Moreover, transformation of E. coli cells with the foreign DNA is easy and requires minimal amount of DNA.

In the prokaryotic hosts like bacteria, bacterial promoters like those of lac or trp genes would have to be used. They are highly inducible in the presence of small amount of certain chemical like IPTG. Some vectors use promoters derived from bacteriophage l, like the PL promoter, which can be induced at high temperature using a temperature-sensitive repressor. Modern-day vectors for bacterial expression systems use synthetic promoters like lac derived from trp, lac, PL etc., which combine the high expression properties of several promoters and are capable of giving several fold higher expression values, as compared to the individual native promoters. As the bacteriado not have the capacity to process introns, the heterologous gene to be expressed should be free from introns. Such a gene is then cloned downstream of the appropriate promoter. For the better purification of expressed proteins, the hetrologous gene is usually cloned in a way, which produces fusion protein. After this it is to be purified by affinity purification and cleaved with a protease to obtain the pure heterologous protein.

Reports were made regarding the over-production of soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system (Zeng et al., 2010). High yielding recombinant Staphylokinase in bacterial expression system was also studied (Mandi et al., 2009). A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus was also reported (Katzke et al., 2010). Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in E. coli were also highlighted (Kyratsous et al., 2009).

Yeast Expression systems

Yeast is considered both as a microorganism and also as eukaryotes and thus having the advantages over the other expression system. The reason for using the yeast is that unlike E. coli, yeast provides advanced protein folding pathways for heterologous proteins, and when yeast signal sequences are used, yeast can secrete correctly folded and processed proteins. Therefore functional and fully folded heterologous proteins can be released into culture media. Unlike mammalian expression systems, yeast can be rapidly grown on simple growth media.

The favored microbial eukaryotes in the development of recombinant DNA technology are Saccharomyces cerevisiae. Other yeast that are used include Schizosaccharomyces pombe, Pichia pastoris, Hansela polymorpha, Kluveromyces lactis and Yarrouvia lipolytica. One of the advantages of using yeast as opposed to bacterial hosts is that proteins are subjected to post translational modification. Moreover, there is usually a higher degree of authenticity with respect to three dimentional confirmation and the immunogenic properties of the protein. Thus, in a situation where the biological properties of the protein are critical, yeast may provide a better product than prokaryotic hosts. If the protein is produced in S. cerevisiae, glycosylation still occurs at the same locations as the human derived protein, but the glycosylation pattern obtained is very different (Moir and Dumais, 1987). To drive target gene expression in yeast, a number of strong constitutive promoters have been used. For example, the promoters for the genes encoding phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD) and alcohol dehydrogenase (ADH1) have all been used to produce target proteins (Cereghino and Cregg, 1999).

Expression of gene in insect cells

Gene expression in insect cells have emerged in the last few years as an attractive choices for the expression of recombinant molecules. Baculovirus expression systems are the most popular of the insect cell expression systems as they can produce large amounts of active proteins. Baculoviruses belong to a large group of circular double stranded DNA viruses which infect only invertebrates, usually insects (Granados and Federici, 1986). The genome is in the range of 90-180 kbp (Ayres et al., 1994).After three to five days of initial infection, cell lysis takes place resulting in death. The nuclear polyhedrosis viruses are a class of baculoviruses that produce occlusion bodies in the nucleus of infected cells. These occlusion bodies consist primarily of single protein, polyhedron, which surrounds the viral particles and protect them from harsh environments. The polyhedron gene is transcribed at very high levels late in the transfection process. Polyhedrin promoter can be used to drive target gene expression. The baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV) has become a popular tool to produce recombinant protein in insect cells (Fraser, 1992). Protein production in baculovirus infected insect cells has the advantage that very high levels of protein can be produced relative to other eukaryotic expression systems (Possee, 1997; Joshi et al, 2000). In this system multiple genes can be expressed from a single virus. The main disadvantages of producing proteins in this way is that the construction and purification of recombinant baculovirus vectors for the expression of target genes in insect cells can take as long as 4-6 weeks, and the cell grow slowly in expensive medium. There is also a chance of contamination. The main disadvantage of this system is that the expression of the foreign protein is controlled by a very late viral promoter and peaks when the cells are dying from the viral infection (Verma et al., 1998).

As far as research is concerned, signal peptide has been designed for improving recombinant protein secretion in the baculovirus expression vector (Sugai and Tsumoto, 2010). Expression of the capsid protein of Chikungunya virus in a baculovirus for serodiagnosis of chikungunya disease were also studied (Cho et al., 2008). Reports were made regarding the development of a prokaryotic-like polycistronic baculovirus expression vector (Chen et al., 2009). Engineering N-glycosylation pathways in the baculovirus-insect cell system have been also done (Jarvis et al., 1998). Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system have been done (Nagy et al., 1990). Reports were made in concerned with expression of functional human glutaminase in baculovirus system (Campos-Sandoval et al., 2007). Baculovirus vectors for gene therapy were also highlighted (Hu, 2006).Recombinant baculoviruses as expression vectors for insect and mammalian cells were also described (Kost et al., 1999).The baculovirus system has been used to express functionally active antibody molecules (Hasemann and Capra, 1990; Zu et al.,. 1990).

Gene expression in mammalian cells

As per as recombinant human protein is concerned, mammalian host cell would be a better system in comparison to bacteria and other microbes. Often media for growths are complex and expensive, and mammalian cells are generally less robust than microbes when large scale fermentation is involved. There may also be difficulties in the processing of the products. Despite these difficulties, many vectors are now available for protein expression in mammalian cells. Vectors utilize selectable markers and have promoters that enable expression of the cloned gene sequence (Reece, 2004). The promoters that are used commonly are based on simian virus (SV40) or cytomegalovirus (CMV). The proteins produced in mammalian system have the best structural and functional features that are usually most close to their cognate native form. It can satisfy the applications, needs or utility as described in the Table 2.

Application, needs and utility of proteins produced by mammalian expression system.

  1. Diagnostic application.
  2. Proteomic and phenomics study.
  3. Cell line development, drug screening, and in vitro model system.
  4. Assay standards.
  5. Functional studies of the protein (in vitro and ex vivo).
  6. Biochemical analyses.
  7. Transgene expression.
  8. Enzyme kinetics.
  9. Therapeutic application.
  10. Physiology and pathology studies.
  11. Protein-protein interaction experiments.
  12. Protein engineering and mutagenesis studies.
  13. Immunogen for antibodies development.
  14. Prophylactic (vaccine) development.
  15. Drug target discovery and validation.

Animproved recombinant mammalian cell expression system for human transforming growth factor-ß2 and -ß3 preparations was studied (Zou, and Sun., 2006). Expression of recombinant human insulin-like growth factor I in mammalian cells was also reported (Bovenberg et al., 1990). Efforts were made regarding the heterologous systems for expression of mammalian sulfotransferases (Veronese et al., 1994).A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells were also highlighted (Kulman et al., 2007).


Protein analysis and purification is aimed at elucidating the structure and function of protein. Protein purification is usually a multistep process. It exploits a wide range of biochemical and biophysical characteristics of the target protein, such as its source, relative concentration, solubility, hydrophobicity and charge. The ideal purification strives to obtain the maximum recovery of the desired protein, with minimal loss of activity, combined with the maximum removal of other contaminating proteins. The methods applied for protein purification must be mild to protect and preserve the native confirmation of the molecule and its bioactivity. One should aim the following as listed in the Table 4 while designing the purification protocol for protein.

Aim during protein purification

  1. High recovery of protein.
  2. To obtain highly purified end products.
  3. Reproducibility within the lab, in other labs and also when either scaled up or down.
  4. Economical use of reagents.
  5. Convenience with regard to time.

The two key parameters used to develop most purification protocols are the chemical structure and physical properties of the protein. Charge density, isoelectric points (PI), PH stability are some of the properties of proteins that can be exploited during purification. A variety of separation techniques are capable of resolving protein on the basis of differences in net charge. These include gel electrophoresis and ion exchange chromatography.

The purification of a bioactive molecule is often accomplished by using a definitive assay designed to recognize a property of protein. Sensitivity is often the limiting factor as the protein is present in extremely small amounts. The assay should be as specific as possible to avoid wasting time and possibly producing an artifactual result. Protein quantification can be performed by the methods listed in the Table 5.

Recombinant proteins may often be difficult to purify and will require multiple time consuming chromatographic steps to be performed before an acceptable level of purity can be achieved. Proteins tags are available which has solved the problem. Protein purification tags are protein sequences that possess high affinity binding properties for particular molecules. The tag allows the target protein to bind to a solid support, usually in the form of a column matrix, to which very few (if any) other proteins are able to bind. The purification of tagged proteins from hosts cells consist of various steps. First the host cell is allowed to lyse followed by the binding of the tagged protein to an affinity column. The column is then to be washed to remove untagged proteins, finally followed by the elution of tagged protein itself. Various tags are available to make the purification of recombinant protein easy.

The His-tag

His-tag is the simplest among all protein purification tags. There are six histidine residues that composed this tag. The DNA which is responsible for coding these residues is cloned into the target gene in such a way that the produced protein contains at some point in its polypeptide sequence, six consecutive histidine residues (Hoffman and Roeder, 1991). At the time of cloning the tag is placed either at the extreme amino or extreme carboxyl-terminal end of the protein, where it is less likely to impair protein function. If the central region of the protein is already recognized to be non essential, then the tag can be placed in the middle of the protein (Zenke et al., 1996). There are certain metal ions with which histidine can bind with high affinity and non-covalently. Metal ions, eg, nickel, are bound to a resin matrix and used to capture his-tagged protein (Yip and Hutchens, 1996). For this purpose the most commonly used resins have nitrocellulose acid (NTA) covalently attached to them. There are four coordination sites in NTA that bind a single nickel ion very tightly. At least six histidine residues are needed to provide the necessary binding affinity to firmly adhere the tagged protein to the column. The other protein will not bind to the column and were eluted out living behind the tagged protein attached to the column. Finally the tagged proteins were removed by changing the concentration of buffer (Reece, 2004).

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using gateway technology have been reported (Halabian et al., 2009). An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in E. coli in a soluble form with an amino-terminal His-tag (Dharmarajan et al., 2009). EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in E. coli, purified and crystallized (Raghunathan et al., 2009).

The GST-tag

Glutathione is a tripeptide made up of glutamic acid, cysteine and glycine. Glutathione-S-transferace (GST) binds to glutathione with high affinity. The gene encoding this protein is fused in the correct reading frame, to the target gene and a fusion protein is produced from an expression vector. Host cells producing the fusion protein are then considered. The cells having the protein are broken and soluble proteins are applied to a column having glutathione (eg glutathione-agarose). The specific interaction between GST and glutathione will result in the binding of the fusion protein to the column. The majority of host proteins are not able to adhere to the column. The protein bonded to the column can then be eluted by washing with a high concentration of glutathione to compete for the interaction with the column. It is desirable then to remove the GST portion of the fusion protein.

To achieve this requirement, a DNA coding for the amino acid sequence of a specific protease cleavge site is to be introduced between the GST and the target gene placed in an expression vector. The purified protein is treated with protease. This will result in generation of two polypeptides. One peptide will be free target protein and other will be GST itself. Target protein can be then separated from GST by applying it back to the glutathione column. The GST will again bind to the column, but the target protein will not. The column flow through can be eluted and will contain the purified target protein. A number of specific protease has been used to cleave target fusion protein obtained by using purification tags. A number of these proteases with recognition and cleavage site have been listed in the Table 6.

Full-length human olfactory receptor (hOR) 2AG1 was over expressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene (Song et al., 2009).

The MBP-tag

E.coli contains malE gene which encodes maltose binding protein (MBP). Maltose is a disaccharide which is made up of two molecules of glucose. MBP is a 40kDa monomeric protein. In an expression vector the target gene is inserted downstream from the malE gene of E. coli that results in the production of an MBP fusion protein (Kellermann and Ferenci, 1982). Purification in one srep of fusion protein is achieved using the affinity of MBP for cross linked amylase (di guan et al., 1988). The target protein is bound to an amylase column and eluted with maltose. The MBP-target fusion is cleaved with a protease and again applied to the amylase column. The protein of interest will not get attached to the column and thus get separated from MBP. Increased solubility of integrin beta A domain using maltose-binding protein as a fusion tag was studied (Lee et al., 2006). Mitochondrial fraction of apoptotic cells contains membrane protein called as p18Bax. High-yield expression and purification of p18 form of Bax as an MBP-fusion protein was also been performed (Eliseev et al., 2004).


IMPACT can be expanded as intein mediated purification with an affinity chitin binding tag. It is also used in protein purification. Inteins are proteins that are found in wide variety of organisms, which excise themselves from a precursor protein and in the process, ligate the flanking protein sequences (Cooper and Stevens, 1995). IMPACT uses the protein self splicing of inteins to remove the purification tag and give pure isolated protein in single chromatographic steps. Most inteins have asparagine at their carboxyl terminal and a cystine residue at their amino terminal end. All the information required for the splicing reaction is stored within the intein itself, and if these sequences are placed in the context of target protein they still splice themselves out. An expression vector is taken to which the target gene is cloned such that a three component fusion protein is obtained, in which a target protein ¾intein¾chitin binding domain fusion protein is produced.

In IMPACT system the E.coli is used and fusion protein is made in the said bacteria. The fusion protein is passed through the chitin column where it binds. Thiol containing compounds such as dithiothreitol (DTT) can be used to cleave off the protein from the column at 4 oC. As the process is slow so overnight incubation is required to complete which is problematic if the target protein is not stable under these conditions. The target protein produced by this method is native except for DTT thioester moiety attached at the carboxyl terminal end. This thioester is unstable. It will hydrolyse to yield native protein.

The expressed enzyme (Uracil-DNA glycosylase from Bacillus sp. HJ171) was purified in one step using the intein mediated purification with an affinity chitin-binding tag purification system (Kim et al.,2008). Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris was also reported (Srinivasa et al., 2008). Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli was also explained (Sharma et al., 2006). Simulation of large-scale production of a soluble recombinant protein expressed in E. coli using an intein-mediated purification system was also reported (Sharma et al., 2005). Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide was also described (Yu et al., 2004).


Tandem affinity purification tag (Tap-tag) is also one of the ways involved in protein purification. The Tap-tag helps in the rapid purification of complexes from a relatively small number of cells without prior information of the complex composition, activity or function (Rigaut et al., 1999; Gavin et al., 2002). This is a two-step purification procedure which is highly specific and can isolate contaminant free protein complexes. At the 31-end of a target gene, the DNA encoding tandem affinity purification tag (Tap -tag) is cloned so that little disruption is made to its ability to be transcribed, and the fusion protein should be produced at the same level as the wild-type target protein. The Tap-tag encodes a calmodulin binding peptide and protein A from Staphylococcus aureus. These two are separated from each other by a TEV protease cleavage site (Puig et al., 2001). Tagged protein containing cells were lysed. It is then applied to a column containing IgG, which binds with greater affinity to protein A. The fusion protein and its associated proteins are removed from the column using TEV protease and then applied directly to a calmodulin bead column, in the presence of Ca2+ and eluted using chelating agent like EDTA.

An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes was reported (Cipak et al., 2009). Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins (Fernández et al., 2009). A modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human was used (Cochrane et al., 2009). Vectors for carrying out TAP-tagging in Candida albicans and a protocol for purification of complexes containing TAP-tagged proteins were presented (Blackwell and Brown., 2009). Tandem affinity purification (TAP)-tagging approaches were also highlighted (Suter et al., 2006). A number of protein tag has been listed in the Table 7.


A number of systems capable of expressing gene of interest have been explained. The methods for purifying proteins have been highlighted. Based on literature survey various tags used in protein purification have been discussed. This article will provide enough knowledge to those researchers who were going to start their carrier in the field of gene expression and protein purification. More effort is required to explore new systems which can express the gene more efficiently. Moreover it is the requirement of the present time to discover the ways of protein purification that can purify protein efficiently and quickly without loosing its biological activities.


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