Inexpensive and Easy: Bacterial Systems Among the In vivo expression systems, bacterial systems produce and purify protein using the least expensive and easiest reagents and equipment. There may be a trade- off in terms of protein function when using a bacterial system. For example, if you need activity that depends on one or more PTMs, consider a yeast or insect system. Functionality and Production: Yeast or Insect Systems Yeast expression systems are easier and less expensive to use than higher eukaryotic cells. They are ideally suited for large-scale production of recombinant proteins. Yeast systems are also amenable to genetic manipulation. However, if specific PTMs are required for activity in functional studies, an insect system may be a better choice. Insect expression systems produce protein that has PTMs similar to mammalian systems and is often properly folded and functional. Insect systems are ideal for producing moderate to high levels of eukaryotic proteins for structure-function assays. Higher Function: Mammalian Systems Mammalian expression systems produce protein in the most native and active form. Select one of these systems to explore protein function in the mammalian cellular environment or to localize your recombinant protein to a specific organelle within the cell.

Bacterial Systems

Most researchers want to obtain the highest level of expression in bacterial cells using the T7 promoter. However, in some cases, having a tightly controlled, modulated system is the only way to obtain expression of a gene of interest. Examples of when you might want a tightly-controlled, modulated system include: Protein is toxic when expressed in bacteria Protein is insoluble If your protein is completely uncharacterized or unlikely to have toxicity and solubility issues, then choose to have a high level of induced expression for maximal protein yield. Optimal Expression

Although some researchers prefer to express native, untagged protein, many researchers put a small epitope tag on their protein. Here are some typical reasons to 'tag' a protein: Easier purification Easier detection when a protein-specific antibody is unavailable Increased solubility If you are interested in tagging your protein, use the following rule of thumb. Use an N-terminal tag if you know that the C-terminus of your protein is most important for function. Use a C-terminal tag if you know that the N-terminus of your protein is most important for function or if you Don't know much about your protein's functional domains. Don't forget, using a vector with a C-terminal tag may be more versatile. You can either clone a gene in frame with the tag to get a tagged recombinant protein or you can clone in a gene with a stop codon to limit translation to your protein only. To Tag or Not to Tag?

T7 Expression Vectors

High-level expression from the bacteriophage T7 promoter T7 gene 10 sequence to provide protein stability N-terminal polyhistidine (6xHis) tag for rapid purification with ProBond® resin N-terminal Xpress® epitope for protein detection with the Anti-Xpress® Antibody Enterokinase cleavage site for removal of fusion tag f1 origin for ssDNA rescue to allow easy sequencing and mutagenesis

His Patch ThioFusion System The His-Patch ThioFusion System has the following features: Recombinant proteins are fused to HP-thioredoxin for increased solubility and rapid purification Expression is driven by the trc promoter and induced to high-levels with IPTG. Expression can be carried out in commonly-used E. coli strains. An enterokinase cleavage site downstream from the HP-thioredoxin gene allows removal of the fusion partner. pThioHis vector is provided in three reading frames for simplified subcloning in frame with the HP-thioredoxin gene. Prepacked ProBond columns and purification buffers are provided to make HP- thioredoxin fusion protein purification fast and easy. A comprehensive manual and trouble-shooting guide to ensure your success.

pL Expression System The pL Expression System offers the tightest transcriptional control of any bacterial expression system. The pL Expression System uses the strong pL promoter to drive expression of the gene of interest. The pL promoter is controlled by the lambda cI repressor protein which is expressed in the E. coli host. The cI repressor gene was engineered into the bacterial chromosome under the control of the tightly-regulated trp promoter. Expression of your gene of interest is induced by the addition of tryptophan. The pL Expression System is so tightly controlled that it can be used to express even highly toxic proteins.

Baculovirus Expression System

Baculovirus Expression System Overview High Level of Protein Expression. Yields of up to 100 mg of protein per 10 9 cells. Post-Translational Modifications. Allows disulfide bond formation, phosphorylation, glycosylation, oligomerization. Relevant Cellular Compartmentalization of Proteins. Secreted, membrane-bound, cytoplasmic or nuclear. Capacity of Large cDNA Inserts. Accommodates genes up to 15 kb. Simultaneous Expression of Multiple Genes. With multiple promoter transfer vectors.

Tet-On & Tet-Off Gene Expression Systems

Precise, dose-dependent regulation High-level induction over extremely low basal expression Proven transgenic & cell culture applications Largest selection of Tet-inducible cell lines Available in retroviral & adenoviral formats

Tet Systems are the gold standard for gene expression in transgenic organisms because of their tight off-state (Figures 2, 5, 6 and 7), well-characterized inducers, and demonstrated ability to limit gene activation to the gene of interest. Proven tissue specificity in vivo (Figure 4), induction through in vivo barriers (Figure 5) and the availability of Tet transgenic mice through Jackson Labs make Tet the clear choice for in vivo induction. The Tet System vector design allows you to drive the transactivator with a tissue-specific promoter (Figure 4), limiting non-specific effects. Dox crosses both the blood-brain (8) and placental barriers, and is transferred across the placenta and through breast milk (Figure 5). These features make the Tet System ideal for expressing embryonic lethal genes, when the presence of the protein of interest during development would have severe developmental implications (3, 8 and 9). Ideal for transgenics

The Tet Systems have an excellent response time to changes in the concentration of doxycycline (Dox) or tetracycline. Changes can be noted in as little as one hour (Figures 6 and 7). Fast kinetics

Mammalian Expression

Vectors for Stable Mammalian Expression High level constitutive transcription from mammalian enhancer promoter sequences plus transcription termination and polyadenlyation signals. Prokaryotic sequences to permit growth and maintenance in E. coli as well as isolation of single stranded DNA. Versatile multiple cloning sites to permit unidirectional or bidirectional cloning. Neomycin, Blasticidin, Hygromycin or Zeocin resistance genes for selection of stable cell lines. pcDNA3.1 pcDNA3.1-E pcDNA4/HisMAX pcDNA4/HisMAX-E pcDNA3.1/Hygro pcDNA3.1/Zeo pZeoSV2 pRc/CMV2 pBudCE4 pRc/RSV Table 1 - Echo ™ -Adapted Expression Vector - Mammalian Echo ™ -Adapted Vector PromoterAdvantage pcDNA3.1-E (Strong, constitutive expression) CMV

Enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus (CMV) for high-level expression Polyadenylation signal and transcription termination sequences from the bovine growth hormone (BGH) gene to enhance mRNA stability SV40 origin for episomal replication and simple vector rescue in cell lines expressing the SV40 large T antigen (e.g. COS1 and COS7) T7 promoter/priming site for in vitro transcription of sense RNA and sequencing of inserts f1 origin for rescue of single-stranded DNA for mutagenesis and sequencing (sense strand) Ampicillin resistance gene and ColE1 origin for selection and maintenance in E. coli

InsectSelect™ Vectors The InsectSelect™ System uses the expression vectors pIB/V5-His, pIZ/V5-His and pIZT/V5-His to produce recombinant proteins either transiently or stably in insect cell lines. The vectors feature the Orgyia pseudotsugata immediate-early 2 (OpIE2) promoter (1) for constitutive expression. The vectors also feature either the Blasticidin or Zeocin™ resistance gene depending on the vector you choose.

Ecdysone-Inducible Expression System Description: The Ecdysone-Inducible Mammalian Expression System is designed for tightly-regulated expression of your gene of interest in mammalian cells. Based on a unique insect mechanism, there is almost no detectable basal expression and greater than 200- fold inducibility in mammalian cells.

Adenoviral vs. Retroviral Expression Systems Adenoviral vs. Retroviral Expession Systems Adenoviral ExpressionRetroviral Expression Episomal gene expressionLong-term, stable gene expression; inheritable Infects dividing & nondividing cellsInfects dividing cells only High-level protein expressionModerate protein expression Two-vector system: pShuttle & Adeno-X Single vector system Viral titers of up to pfu/mlViral titers of up to 10 6 cfu/ml (Can be concentrated to 10 9 cfu/ml) Accommodates inserts of up to 8 kbAccommodates inserts of up to 6.5 kb Elicits immune reactions in vivoDoes not elicit immune reactions in vivo

BD Adeno-X™ Expression System Generate recombinant adenovirus for expressing a protein in only two weeks High protein expression levels. Adenoviral-mediated gene transfer provides high protein expression levels in mammalian cells (1). Because recombinant adenovirus containing your gene of interest infects target cells with multiple copies and high efficiency, cells transiently express the protein of interest at very high levels—up to % of total protein. Efficient infection of many mammalian cell types. Adenoviral particles efficiently infect a majority of human and many nonhuman cell types including mouse, rat, dog, chicken, rabbit, sheep, pig, and nonhuman primates (2–11). Adenovirus infects both dividing and nondividing cells. This wide variety of target cells combined with high-level gene expression makes the BD Adeno-X™ System ideal for a number of research applications, including gene therapy development, gene function analysis, antisense therapy, vaccine development, and some transgenic animal studies. Optimized protocol for fast results. Our protocol has been optimized so that recombinant adenovirus can be generated in 10–17 days. Instead of homologous recombination, this protocol uses an efficient, ligationbased method with pre-linearized, ready-to-ligate BD Adeno-X™ Viral DNA (Figure 2). The system also provides a convenient shuttle vector, pShuttle2, and a unique double digestion buffer that saves time during cloning. Simple cloning procedures. By using a ligation-based approach, the Adeno-X System eliminates nonrecombinant adenovirus. With our optimized protocol, this in vitro ligation approach produces recombinant adenovirus much more rapidly and reliably than conventional homologous recombination approaches (Table I).