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Date: February 2, 1998 Chart 1 Chart 2 Prokaryotic expression systems, reviewed in the September 1, 1997, issue of The Scientist were part of the early repertoire of research tools in molecular biology. Although the expression of recombinant protein in prokaryotes provided a means to develop other research tools (antibodies, for example ) and study basic aspects of biological function, the scope and depth of this research were limited, especially with regard to eukaryotic proteins. The de novo synthesis of recombinant eukaryotic proteins in a prokaryotic system imposed a number of detrimental constraints on the eukaryotic gene product. Among the two most critical were improper protein folding and assembly, and the lack of posttranslational modification, principally glycosylation and phosphorylation. Prokaryotic systems do not possess all the appropriate protein synthesizing machinery to produce a structural and/or catalytically functional eukaryotic protein. To overcome this hurdle, a new technology was developed that utilized eukaryotic cells and the natural parasites of eukaryotic cells; viruses. Viral infection is basically the redirection of a host cell's transcriptional and translational machinery toward synthesis of viral-specific products. Why not engineer the virus (vector) to redirect synthesis of a desired gene product (recombinant protein) instead of undesired viral products? The premise of this technology was novel in its simplicity: let nature work for us! Utilizing the eukaryotic cell's protein synthesizing machinery and expression vectors containing various transcriptional elements (strong promoters) derived from viruses, a researcher can now engineer a cell to produce native eukaryotic protein. Eukaryotic expression vectors represent the newest genre of science discovery tools in biotechnology and have been instrumental in advancing basic protein science, especially in the area of structure-function relationships and in clinical studies on the therapeutic application of the expression of foreign proteins in diseased cells. Eukaryotic expression vectors are of two basic types: virion or virion-plasmid hybrids. Virion-type vectors are most commonly used for the delivery of foreign genes, or a replacement for a defective host gene, into mammalian cell hosts. Thus, they are prominent in medical gene therapy research. One of many examples of viral gene therapy is the adenovirus vector-mediated delivery of the cystic fibrosis (CF) transmembrane conductance regulatory gene into nasal epithelia of human patients with CF and the subsequent transient relief of the CF chloride transport defect (J. Zabner et al., Cell 75:207-216, 1993). For an exhaustive review of viral vector applications in this area, see Viral Vectors: Gene Therapy and Neuroscience Applications (M.G. Kaplitt and A.D. Loewy, eds., Academic Press, San Diego, CA, 1995). The other type, virion-plasmid hybrid vectors, are customarily used to facilitate the over-expression of protein that mimics the native form in all respects: structure, catalytic activity, immunological reactivity, and biological function, something that is difficult, if not impossible, to achieve using prokaryotic expression systems. Hybrid vectors have been used extensively to produce copious amounts of recombinant and native protein. The availability of authentic protein has hastened the development of structure-dependent epitope-specific antibodies to native proteins, the crystallization and subsequent X-ray/NMR analysis of proteins in their native, correctly folded, posttranslationally modified state, the characterization of gene products for genes whose phenotype may or may not be known, and the isolation of proteins whose native form is in such low abundance that they are very difficult to purify from the original natural organism or tissue. In addition, eukaryotic expression systems have provided new avenues to study regulation of cloned eukaryotic genes in defined genetic environments and to analyze transcript posttranscriptional and polypeptide post-translational processing, events that were impossible to study in prokaryotic systems. This sketch focuses on the hybrid vectors and in particular those that are commercially available for the research community. Keynote features of all eukaryotic expression vectors include various sequence elements that explicitly define the level of expression, the transcriptional start and stop points, postprocessing (transcript splicing, poly-adenylation, and so forth.) transport, selectable markers, and in some cases a peptide tag to facilitate isolation and purification of the gene product. One of the earliest eukaryotic expression vectors, pSV2, is a composite of sequence elements from the papova virus, Simian Virus 40 (SV40; see S. Subramani and P.J. Southern, Analytical Biochemistry, 135:1-15, 1983), and the prokaryotic cloning vector, pBR322. SV40 sequence provided transcriptional enhancers, promoter (early region), splicing signal (small t-antigen gene), and the polyadenylation signal element, and pBR322 provided the origin of replication (ori) element. The presence of a selectable marker, pBR322 AmpR gene, completes this model eukaryotic expression vector. Practically all modern eukaryotic expression vectors possess one or more of these elements. Although the SV40 transcriptional elements were the first and most popular, transcriptional elements from other viruses (i.e., cytomegalovirus [CMV], Murine sarcoma virus [MSV], Rous sarcoma virus [RSV], Mouse mammary tumor virus [MMTV], and Semliki Forest Virus [SFV]) are also coming into widespread use. Currently, all eukaryotic expression systems (virion and hybrid) can be grouped into four distinct classes based upon host type: yeast, drosophila, insect (nondrosophila), and mammalian. This product synopsis focuses on hybrid vector systems for mammalian cell hosts. Another synopsis to appear in a later LabConsumer issue (November 9, 1998) will emphasize eukaryotic (hybrid) expression systems for yeast, drosophila, and insect cell hosts. in Mammalian Cells Some of the major differences in the various mammalian eukaryotic expression vectors available today center around the type of promoters (SV40, CMV, human EF1alpha, MMTV-LTR, MSV-LTR, RSV-LTR), expression (constitutive, inducible) and mechanism of induction, selectable markers (antibiotic or drug resistance and/or co-expression of easily identifiable proteins), and recently, elements for subcellular (mitochondria, nucleus) targeting of the gene product. See Table 1 and 2 for an overview of vectors, vector properties, and their respective corporate suppliers. Generally, recombinant proteins are expressed in a constitutive manner in most eukaryotic expression systems. The addition of inducible promoters or enhancer elements into the vectors has created a new family of research tools tools that provide new opportunities to analyze temporal, spatial, and inducer-dependent gene expression and provide an alternative mechanism to produce recombinant proteins. Inducible promoters include heat shock protein, metallothionien, and human and mouse growth hormone, MMTV-LTR, while inducible enhancer elements include those for ecdysone, muristerone A, and tetracycline/ doxycycline. The Tet-On and Tet-Off Gene Expression Systems (CLONTECH) use a tetracycline (Tc) responsive element to maintain recombinant protein expression in an on (constitutively off, induce with Tc) or off (constitutively on, repress with Tc or doxycycline) mode. The Ecdysone-Inducible Mammalian Expression System (Invitrogen) comprises a vector (pIND) with a hybrid ecdysone-glucocorticoid responsive element (E/GRE) directly upstream from its promoter (located in front of the target gene insertion site), and a helper plasmid (pVgRXR) that encodes a modified (containing a VP16 transactivation domain) ecdysone (VgEcR) receptor and retinoid X-receptor (RXR) proteins. Host cells cotransfected with pIND and pVgRXR and expressing housekeeping levels of the VgEcR and RXR receptor proteins can be induced to overexpress target gene protein with muristerone A. Induction is mediated by the binding of muristerone A to VgEcR and subsequent association with RXR. This protein complex then recognizes the E/GRE enhancer and binds, and the VP16 transactivation domain then interacts with the vector promoter initiating target gene expression. Invitrogen has also added a new twist to this burgeoning field with the introduction of a vector (pShooter) containing signal elements that target the expressed gene product to specific subcellualr locations. Selectable markers generally fall into two functional categories: recessive and dominant. The recessive markers are usually genes that encode products that are not produced in the host cells (cells that lack the "marker" product or function). Marker genes for thymidine kinase (TK), dihydrofolate reductase (DHFR), adenine phosphoribosyl transferase (APRT), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) are in this category. Dominant markers include genes that encode products that confer resistance to growth-suppressing compounds (antibiotics, drugs) and/or permit growth of the host cells in metabolically restrictive environments. Commonly used markers within this category include a mutant DHFR gene that confers resistance to methotrexate; the gpt gene for xanthine-guanine phosphoribosyl transferase, which permits host cell growth in mycophenolic acid/xanthine containing media; and the neo gene for aminoglycoside 3'-phosphotransferase, which can confer resistance to G418, gentamycin, kanamycin, and neomycin. Practically all eukaryotic expression vectors used today possess at least one marker from either or both of these categories, although dominant markers are the most common. Recent additions to the selectable marker inventory include resistance to Zeocin (pcDNA3.1, pIND, pZeoSV2, pTracer vectors [Invitrogen]) and Hygromycin (pcDNA3.1, pREP [Invitrogen]; IRES [CLONTECH] vectors) and the co-expression of green fluorescent protein (visible marker) with pTracer (Invitrogen) and Adeno Quest (Quantum Biotechnol-ogies). The use of autofluorescent proteins is unique here because it will provide a mechanism to visually asses if host cells are overexpressing recombinant protein. Host-range specificity for virion vectors is determined primarily by the presence of recognizable host cell surface receptors, whereas for virion-plasmid hybrid vectors, the determinant is the degree to which the host's cellular machinery recognizes the transcriptional control signals of the hybrid vector. Transcriptional enhancers appear to be the primary determinants of cell-type specificity. After the target gene has been engineered into the appropriate cloning vector, the vector will need to be introduced into the host cell. Given the infectious nature of the viruses from which most eukaryotic expression vectors are derived, it would seem that the introduction of the vector into the host would be rather straightforward. Unfortunately, this process can be complicated. Numerous protocols utilizing different chemistries and host cell membrane characteristics may be drawn upon to accomplish this task. The introduction of the vector into mammalian cells can be effected by microinjection, electroporation, and calcium phosphate-, DEAE-dextran-, polybrene-, DMSO-, or cationic lipid-mediated transfection. Although the method of choice usually depends on the cell type and cloning application, cationic lipids or liposome-mediated transfection protocols generally yield the highest and most consistent transfection efficiencies in mammalian cell systems. Thus, many corporate suppliers of expression systems recommend a lipid-based transfection protocol, with many providing proprietary liposome reagents (for example, CLONTECH's CLONfectin Transfection Reagent, Invitrogen's PerFect Transfection Kit, Promega's Transfectin and Tfx-50 reagents, or Life Technologies Lipofectin Reagent). For a more detailed overview of transfection protocols, please refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, N.Y., 1989. The search for vectors that will permit inclusion of increasingly larger target DNA, novel gene induction modes, marker systems, and new hosts has provided considerable impetus behind the development of new vectors, c.f., the numbers of new vector products and companies that have appeared on the market the last five years. The newest product mentioned in this sketch became available less than three months ago. The rapid advent of new expression products surely warrants the watchful eye of the researcher, and the LabConsumer aims to inform you of these products as soon as they are introduced into the marketplace. Chart 1 Chart 2 Christopher Smith is an Associate Staff Scientist at San Diego Super Computer Center and can be reached at csmith@sdsc.com

Creative Expression: Mammalian Expression Vectors and Systems

Feb 1, 1998

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