Expression Vectors 5221 Plasmid vectors

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5.2.2.1a Plasmid structure

A wide selection of plasmid-based expression vectors is now available. For many studies of gene expression, small quantities of protein are sufficient and can be obtained using transient assay systems. When larger amounts of protein are required, it is necessary to identify clonal cell lines in which the vector sequences are retained during cellular proliferation ('stable' cell lines). This can be achieved either by episomal plasmid replication or by the integration of the vector into the host cell genome. Cell lines containing foreign deoxyribonucleic acid (DNA) can be identified by the use of a suitable selectable marker gene (Gorman 1985; Chisholm 1995). The choice of vector will depend upon the host cell employed and the amount of protein required. Each vector must contain bacterial 'backbone': sequences that allow replication and maintenance in bacteria, a translation initiation sequence, a promoter/enhancer sequence and a polyadenylation signal. If stable expression is needed, a selectable marker gene is required, while episomal plasmid replication is directed by a variety of viral elements. Optional components may include the presence of an antigen epitope that permits detection/visualization of expressed gene products and a purification tag. A reporter gene, whose expression can give quantitative indications of transfection or transcriptional activity, can be added if required. The read-out from this can be either enzymatic, such as chloramphenicol acetyl transferase, or bioluminescent, such as firefly luciferase or green fluorescent protein (Alam & Cook 1990; Chisholm 1995).

The bacterial backbone always includes an origin of replication (ori) and its associated cis-acting control elements, the whole genetic unit being termed a replicon. Replicons derived from the pMB1 (or ColE1) plasmid do not require plasmid-encoded functions for replication, and can function in the absence of ongoing protein synthesis, leading to the accumulation of several thousand copies of the plasmid in the cell (Stadenbauer 1978). One or more antibiotic resistance genes (i.e; ampr-ampicillin, kanr-kanamycin, tetr-tetracycline or camr-chloramphenicol) for selection in E.coli must also be present. Inclusion of elements of the lacZ operon permits blue/white screening of recombinant plasmids, while a multiple cloning site (MCS) with a wide range of single-cutting RE sites for insertion of the gene to be expressed is essential. The MCS can be designed to permit the cloning of blunt or sticky-ended polymerase chain-reaction-derived cDNA and to allow insertion in a specific orientation. Other genes that may be present include a variety of suppressor transfer ribonucleic acid (tRNA) genes to inhibit bacterial nonsense alleles, a f1 or M13

poly A MCS

poly A MCS

Pee14 Vector

Map of plasmid pEE14, an expression vector for CHO cells. GS= hamster glutamine sythetase minigene (contains a single GS intron expressed from the SV40 late promoter (SV40l); hCMV-MIE= human cytomegalovirus major intermediate early enhancer-promoter; MCS = multiple cloning site; Apr= ampicillin resistance gene; poly A = polyadenylation signal

Plasmide Pee14

Map of plasmid pUC18, a general bacterial cloning vector. ori = origin of replication; Apr = ampicillin resistance gene; lacI =lac repressor gene; lac Z' = lacZ' gene; Plac = lac promoter; MCS = multiple cloning site

Map of plasmid pEE14, an expression vector for CHO cells. GS= hamster glutamine sythetase minigene (contains a single GS intron expressed from the SV40 late promoter (SV40l); hCMV-MIE= human cytomegalovirus major intermediate early enhancer-promoter; MCS = multiple cloning site; Apr= ampicillin resistance gene; poly A = polyadenylation signal

Map of plasmid pUC18, a general bacterial cloning vector. ori = origin of replication; Apr = ampicillin resistance gene; lacI =lac repressor gene; lac Z' = lacZ' gene; Plac = lac promoter; MCS = multiple cloning site

Figure 5.2 Example of a general cloning vector (pUC18) and mammalian expression vector (pEE14).

ori to generate single-stranded DNA templates for sequencing or site-directed mutagenesis, and promoter sequences for T3 or T7 bacteriophage DNA-dependent RNA polymerase to permit the generation of RNA transcripts.

To ensure that the inserted gene is optimally expressed, a 'Kozak' translational initiation sequence (CCA/GCATG) is nearly always included before the initiating ATG of cDNA gene expression constructs (Kozak 1986). Polyadenylation of messenger ribonucleic acid (mRNA) is critical for message stability and transport from nucleus to ribosome. Most expression vectors contain either the SV40 early or bovine growth hormone polyadenylation signal to allow for this (Goodwin & Rottman 1992; Kaufman & Sharp 1982). Examples of a basic cloning plasmid (pUC18) and a mammalian expression plasmid (pEE14) are given in Figure 5.2.

5.2.2.1b Selection

Selectable markers are either genes (mostly bacterial), which establish drug resistance in cell culture, or which are dependent on specific mammalian cell genotypes (Table 5.1). The first category of markers is commonly represented in commercial vectors by the bacterial aminoglycoside phos-photransferase gene aph, which detoxifies the protein synthesis inhibitor drug G418 (neomycin/ geneticin)(Colbere-Garapin et al. 1981) hygromycin-B-phosphotransferase (hph) which inhibits hygromycin-B (Blochlinger & Diggelmann 1984) and the ble, blc and pac genes that confer resistance to the antibiotics bleomycin (zeocin), blasticidin and puromycin respectively (Vara et al. 1986; Mulsant et al. 1988). Many of the second category of genes are 'amplifiable', and will be discussed in relation to strategies that enhance protein expression (Bebbington 1995).

5.2.2.1c Promoters

In many cases, maximal amounts of biologically active, stable recombinant protein are required. The inclusion of specific elements within an expression vector can make the difference between poor and high yields of protein from a given cell line. The first essential choice is that of the promoter/enhancer sequence. When not working with a toxic gene, timing of expression is less important and a constitutively expressing promoter can be used (see below). However, if the gene product is toxic or unstable, expression can be controlled by the use of an inducible promoter

GENE EXPRESSION Table 5.1 Genes (and their specific inhibitors) used for selection of clonal cell lines.

Gene Product Inhibitor Reference

Bacterial/fungal selectable markers

Neo

Aminoglycoside

G418 (geneticin/neomycin)

Colbere-Garapin et al.

phosphotransferase

(1981)

Hph

Hygromycin-B-

Hygromycin B

Blochlinger and

phosphotransferase

Diggelmann (1984)

Sh ble

High affinity antibiotic binding

Bleomycin (zeocin)

Mulsant et al. (1988)

protein

Bsd

High affinity antibiotic binding

Blasticidin

Mulsant et al. (1988)

protein

Pac

Puromycin N-acetyltransferase

Puromycin

Vara et al. (1986)

trpB

Tryptophan synthetase

Indole

Hartman and Mulligan

(1988)

hisD

Histidinol dehydrogenase

Histidinol

Mammalian cellular genotypes

Dhfr

Dihydrofolate reductase

Methotrexate

Gs

Glutamine synthetase

L-Methionine sulfoximine

Bebbington (1995)

Ada

Adenosine deaminase

Deoxycoformycin

Asns

Asparagine synthetase

Albizzin

Cad

Aspartate transcarbamylase

W-(phosphonacetyl)-L-

aspartate

Polr2K

Metallothionein-I

Heavy metals

Odc

Ornithine decarboxylase

a-Difluoromethyl ornithine

mdrI

P-Glycoprotein 170

Adriamycin

Rnr

Ribonucleotide reductase

Hydroxyurea

TK

Thymidine kinase (defective)

Xgprt

Xanthine-guanine

Mycophenolic acid

phosphoribosyl transferase phosphoribosyl transferase

(see 5.2.2.1d). The promoter consists of a DNA sequon where RNA polymerase II transcription is initiated, and is positioned so as to direct transcription in a defined direction. Enhancer motifs augment transcription from a promoter and are essential for the highest levels of transcriptional activation.

The most generic and versatile promoter/enhancer assemblies for mammalian expression are derived from viral systems. The human cytomegalovirus major immediate-early viral promoter/ enhancer (hCMV), the Rous sarcoma virus long terminal repeat promoter (RSV-LTR), the simian virus early promoter/enhancer (SV40), and the human elongation factor 1a-subunit promoter are often employed in commercially available vectors (Berg 1981; Gorman et al. 1982; Foeck-ing & Hofstetter 1986; Kim et al. 1990). hCMV is a stronger promoter than either RSV-LTR or SV40 in most cell lines and is the most commonly used. It is particularly effective in adenovirus-transformed cell lines, such as HEK-293 (Gorman et al. 1989). The RSV-LTR is derived from an avian virus, and works best in avian cell lines, while SV40 works well in most cell lines but performs best in lines containing the stably integrated SV40 large T antigen, such as the African green monkey kidney COS cell lines (Berg 1981; Gorman et al. 1982).

5.2.2.1d Enhanced expression

Regulated (or inducible) transcription allows the precise expression of the gene of interest and is often used when the protein product is cytotoxic or cytostatic. Simple inducible promoters are based upon such elements as metallothionein or heat-shock promoters, but lack the precision achievable with engineered systems where the regulatory components are stably integrated into the host cell. These have now reached a considerable degree of sophistication and the choice of a suitable vector can allow the low basal expression of a highly toxic protein, a high induced level of expression for maximum production or modulated expression for functional analysis. Current commercial systems allowing high inducible levels of expression are mostly based on bacterial regulated promoters, such as the modified E.coli lac operon system available from Stratagene (Wyborski et al. 1996); the 'tet-On'/'tet-Off' system based on elements of the Tn-10 encoded tetracycline resistance operon, and the HSV virion protein 16, marketed by Clontech (Gossen & Bujard 1992); and the T-Rex (tetracycline-regulated-expression) Invitrogen system that also employs elements from the E.coli tetracycline Tn-10 suppression mechanism to regulate expression from the hCMV promoter (Yao et al. 1998). Other promoters are regulated by steroid hormones, such as the ecdysone-inducible system based on a Drosophila regulatory mechanism from Invitrogen (No et al. 1996). Dual-regulated systems using both tetracycline and streptogramin are under development (Fussenegger 2001; Fussenegger et al. 2000). The Invitrogen Geneswitch system uses elements from the yeast Gal4 and adenoviral E1b promoters and remains transcriptionally silent until activated by a regulatory protein consisting of the Gal4 DNA binding domain, a truncated human progesterone receptor ligand binding domain, and the NFkB transcription factor p65. This highly controlled system permits the expression of toxic proteins (Wang et al. 1994).

Recent advances include the development of multicistronic expression strategies which permit multigene expression using a single vector to transduce suitable mammalian cell lines. At the most basic level, this allows the expression of product and marker gene from the same plasmid, the most stable coupling being achieved when both genes are expressed from the same promoter. Coordinated expression of the paired genes can be achieved by gene fusions or differential splicing (Kromer et al. 1997; Sonenberg 1994; Attal et al. 1999), but most multigene systems take advantage of an internal ribosomal entry site (IRES) to direct the translation of the second cistron (Fussenegger et al. 1999). Most IRES elements are drawn from poliovirus or encephalomyocardi-tis virus, IRES-mediated translation efficiency varies greatly between the different IRES elements and also with the cell line used (Fussenegger et al. 1999; Borman et al. 1997). IRES-based vectors containing three or four cistrons have been developed and applied to a number of novel technologies including the coupled expression of multicomponent/multisubunit proteins; the cloning of high-producer cell lines by the introduction of a selection marker into the last cistron; one-step regulated gene expression systems using positive-feedback circuits; and multigene metabolic engineering of continuous cell lines using sense, antisense or ribosome technology (Fussenegger et al. 1998; Burger et al. 1999; Lucas et al. 1996). Their use as nucleic acid vaccines is also under investigation (Fussenegger et al. 1999).

Enhanced production in transient assay systems in many cell lines can be achieved by the epi-somal replication of the expression plasmid to high copy numbers. This is usually induced by the presence of the SV40 large T antigen or the Epstein-Barr virus origin of replication (oriP) and nuclear antigen (EBNA-1). Indeed, the level of expression of several proteins in a HEK-293 EBNA line approached that seen with stable cell lines (Meissner et al. 2001) and in a fraction of the time. A complete system based on this 'Mass Transient' technology is now available ('Freestyle 293 Expression System', Invitrogen).

5.2.2.1e Gene amplification

Although the combination of a strong promoter/enhancer, suitable selectable marker and a permissive cell line may lead to increased gene expression from an integrated vector, it is known that the chromosomal location where integration occurs has a profound effect on transcription levels. This so-called 'position effect' can be overcome by increasing vector copy

Cho Protein Production

Transfect CHO/BHK cells

Figure 5.3 Gene amplification.

Assay protein Assay protein production - production -

select lines select lines

Transfect CHO/BHK cells

Figure 5.3 Gene amplification.

number, but other approaches exist as described in Chapter 8. This is done by selection for gene amplification.

When cell lines are cultured in the presence of certain toxic drugs, mutant drug-resistant lines can be isolated. These often arise from the overproduction of an essential enzyme the drug inhibits, the overproduction arising from increased mRNA levels ultimately stemming from increased copy number of the enzyme gene. About a dozen genes have been found to amplify in this way and, if cloned and reintroduced into a cell line that lacked endogenous drug resistance activity, the cloned gene was found to be capable of amplification as well (Table 5.1). The significant feature of gene amplification is that regions of chromosomal DNA adjacent to the enzyme gene are also amplified, hence other sequences on an integrated vector containing the enzyme gene will be co-amplified. This provides a means of progressively increasing mRNA transcription by selecting lines in the presence of increasing concentrations of the specific enzyme inhibitor, and has led to a class of vectors with the most efficient expression characteristics currently available (Figure 5.3). The two most commonly used amplifiable systems (gene/selective inhibitor/cell line) are based on dihydrofolate reductase (dhfr) + methotrexate in dhfr ~ CHO lines, and glutamine synthetase (GS) + methionine sulfoximine in CHO-K1 or murine myeloma NS0 cells (Bebbington et al. 1992). Amplifiable genes and their inhibitors are listed in Table 5.1. Both systems have been used extensively to express recombinant proteins (Page et al. 1991; Rhodes et al. 1994; Bebbington 1995; Jeffs et al. 1996).

5.2.2.1f Affinity fusion partner technology

Genetic fusions comprise a method whereby desired properties from the fusion partner can be added to the target protein. The commonest use is to add an affinity fusion partner to permit purification by immunoaffinity chromatography. A cleavable link can be engineered between the target protein and the fusion partner. The most widely used approach consists of the addition of six histidine residues to the N- or C-terminal of the target protein to permit its capture by solid phases incorporating nickel or cobalt ions (see Section 18.5.6). These 'hexahis' tags are very efficient at extracting monomeric, minimally glycosylated proteins, but are less useful for oligomeric glyco-proteins (Jeffs personal observations). Other fusion partners commercially available are based on maltose binding protein, calmodulin, cellulose binding domain, glutathione ^-transferase, HSV

glycoprotein D, 5"-protein and T7 gene 10. These are extremely useful when no high affinity protein-specific antibodies are available for purification (Ford et al. 1991; Nilsson et al. 1996; Hearn & Acosta 2001).

Other applications of genetic fusions may be employed to enhance the secretion of the target protein from the cell into the ambient medium by use of a secretion signal sequence, or to increase the overall solubility of the chimaeric protein by fusion to a highly soluble partner. Targeting of chimaeric proteins can be achieved by the addition of molecules capable of binding cell surface receptors or polysaccharides, while immunopotentiation of protein antigens in vaccines may be achieved by addition of carrier proteins fused to the immunogen subunit (reviewed by Liljeqvist & Stahl 1999).

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Responses

  • tesfay
    How do pee14 plasmid integrate into cho cells?
    7 years ago
  • carmine
    Where to buy pEE14 vector?
    5 years ago

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