List of some commonly used plasmids for improving expression levels and solubility of recombinant proteins in
Abstract
Plasmids are important vectors for the transfer of genetic material among microbes. The transfer of plasmids causes transmission of genes involved in pathogenesis and survival, to the host bacteria leading to their evolution and adaptation to diverse environmental conditions. A large number of plasmids of varying sizes have been discovered and isolated from various microorganisms. Plasmids are also valuable tools to genetically manipulate microbes for various purposes including production of recombinant proteins. Escherichia coli is the most preferred microbe for production of recombinant proteins, due to rapid growth rate, cost-effectiveness, high yield of the recombinant proteins and easy scale-up process. Several plasmids have been designed to optimize the expression of heterologous proteins in E. coli. In order to circumvent the issues of protein refolding, the codon usage in E. coli, the absence of post-translational modifications, such as glycosylation and low recovery of functionally active recombinant proteins, various plasmids have been designed and constructed. This chapter summarizes the recent technological advancements that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies.
Keywords
- plasmid
- recombinant protein
- promoter
- codon usage
- molecular chaperones
- fusion tags
1. Introduction
Plasmids are defined as extrachromosomal double-stranded circular DNAs within a cell that have the capability to replicate independently of chromosomal DNA. Plasmids are found in many microorganisms including bacteria, archaea and some eukaryotes such as yeast [1]. The advent of new DNA sequencing technologies has successfully determined the complete sequence of 4602 plasmids; most of these plasmids, that is, 4418 are from bacteria, 137 plasmids are identified from archaea and 47 plasmids are identified from eukaryotes [2]. The size of plasmids can vary between 1 and 200 kb, and they more often harbour genes encoding proteins that confer selective advantage to host cells under adverse conditions. Some of the genes are known as resistance genes which confer resistance to certain antibiotics. Genes involved in synthesis of antibiotics and various kinds of toxins are also localized on plasmids. Some of the genes present on plasmids encode for various virulence factors that assist microbes to colonize host and escape from its defence mechanisms. Plasmids also harbour genes that empower bacteria to fix nitrogen.
Plasmids can be present in a single bacterial cell in varying number which may range from one to few hundreds. The usual number of plasmids that are present in an individual cell is termed as copy number and is governed by the size of plasmid and the regulation of replication initiation. Large-size plasmids are present in low copy number and exist as one or very few copies in single bacterium. Such single-copy plasmids employ parABS and parMRC systems, termed as partition system, to equally segregate a copy of plasmid to each daughter cell upon cell division [3].
Independent replication of plasmids requires the presence of a region of DNA that can serve as an origin of replication. A self-replicating unit is termed as a replicon. A classical bacterial replicon comprises of gene for plasmid-specific replication initiation protein (Rep), DnaA boxes, AT-rich region and repeating units called iterons [4]. Large-size plasmids also harbour genes required for their replication, while smaller plasmids employ host replicative machinery to undergo replication.
Plasmids can be transmitted from one bacteria to another by the process of conjugation, and it has been reported that approximately 14% of the currently known plasmids are conjugative [5, 6]. Conjugation is a very efficient mechanism to transfer genes among microbes and thus facilitate the rapid evolution and adaptation of microbes to various adverse environmental conditions [7]. This transfer of genes among bacteria is one mechanism of horizontal gene transfer and is responsible for spread of antibiotic resistance among pathogenic microbes [8, 9].
Plasmids are very commonly used as vectors in the field of genetic engineering for the purpose of cloning and expression of desired genes. Various types of plasmids are now available commercially for cloning and expression of foreign genes in a wide variety of host including
2. E. coli as an expression system for production of recombinant proteins
3. Promoter
Promoter is a very critical region in plasmid vectors, used for the expression of heterologous proteins. Promoter is a stretch of DNA that is involved in the initiation of transcription of a gene and is located upstream of the transcription initiation site of gene. Promoters are normally 100–1000 base pairs in size. In
Plasmid | Promoter | Origin of replication | Antibiotic selection marker | Fusion tags | Size of the fusion tag | Protease cleavage site | Vendor |
---|---|---|---|---|---|---|---|
pGEX-2 T | tac | pBR322 | Ampicillin | GST | 26 kDa | Thr | Pharmacia |
pGEX-3X | tac | pBR322 | Ampicillin | GST | 26 kDa | Xa | Pharmacia |
pGEX-6P | tac | pBR322 | Ampicillin | GST | 26 kDa | Pre | Pharmacia |
pMAL-c2X | tac | ColE1 | Ampicillin | MBP | 42 kDa | Xa | New England Biolabs |
pMAL-c2E | tac | ColE1 | Ampicillin | MBP | 42 kDa | EK | New England Biolabs |
pQE-30/31/32 | T5-lac | ColE1 | Ampicillin | N-6XHis | 1 kDa | None | Qiagen |
pTrxFus | PL | ColE1 | Ampicillin | TRX | 11.6 kDa | EK | Invitrogen |
pET-14b | T7 | pBR322 | Ampicillin | N-6XHis | 1 kDa | Thr | Novagen |
pET-19b | T7-lac | pBR322 | Ampicillin | N-6XHis | 1 kDa | EK | Novagen |
pET-29a-c(+) | T7-lac | pBR322 | Kanamycin | C-6XHis | 1 kDa | Thr | Novagen |
pET-41a-c(+) | T7-lac | pBR322 | Kanamycin | GST | 26 kDa | Thr | Novagen |
pET-43a-c(+) | T7-lac | pBR322 | Kanamycin | NusA | 55 kDa | Thr | Novagen |
pET-SUMO | T7 | pBR322 | Kanamycin | SUMO | 11 kDa | SUMO protease | Invitrogen |
4. Ribosomal binding site
Ribosomal binding site is a very crucial component in plasmids commonly used for expression of recombinant proteins in
5. Codon usage and plasmid containing tRNA genes cognate to the rare codons
Codon usage is a major issue while expressing heterologous proteins, particularly human proteins in
Various strategies have been designed to circumvent the problem of codon bias in
6. Plasmids carrying molecular chaperones for optimization of protein folding
Production of recombinant proteins for therapeutic purposes or various functional studies requires a robust and cost-effective expression system which can synthesize heterologous proteins in soluble form. Although
Plasmid | Chaperones | Promoter | Antibiotic marker | Inducer | Vendor |
---|---|---|---|---|---|
pGro7 | Chloramphenicol | L-Arabinose | TaKaRa | ||
pKJE7 | Chloramphenicol | L-Arabinose | TaKaRa | ||
pG-KJE8 | Chloramphenicol | L-Arabinose Tetracycline | TaKaRa | ||
pTf16 | Trigger factor (tig) | Chloramphenicol | L-Arabinose | TaKaRa | |
pG-Tf2 | Chloramphenicol | Tetracycline | TaKaRa | ||
pBB540 | grpE, clpB | Lac | Chloramphenicol | IPTG | Addgene |
It has been demonstrated that the periplasm of
7. Use of plasmids containing fusion tags to improve solubility
Another strategy to improve the solubility of recombinant proteins is to construct a fusion with a highly soluble protein. Several plasmid vectors are commercially available that carry fusion protein tags. Fusion tags technology can be used to increase protein expression, improve solubility as well as facilitate purification of recombinant proteins. Fusion tags are currently one of the most preferred methods to produce difficult-to-express heterologous proteins in
8. Future perspectives
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