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Tightly Controlled Bacterial Protein Expression - u-szeged.hu

PBAD Expression System Tightly Controlled Bacterial Protein Expression The pBAD system offers: Tightly regulated Expression Dose-dependent induction High Protein yields Simplified detection and purification of expressed Protein Controlled Expression for maximum yields Take control of Expression in E. coli with the pBAD Expression System. Tight regulation allows you to turn Expression on or off. Dose-dependent induction lets you easily modulate Expression levels. No other Bacterial Expression system gives you such control. The pBAD Expression System helps you overcome two of In both cases, a Tightly regulated Expression system is the most common problems for heterologous Protein critical for maximizing recombinant Protein yields.

pBAD Expression System TOPO Represents covalently bound topoisomerase I The pBAD TOPO® TA Expression Kit specifically is designed for one-step cloning and regulated expres-sion in E. coli.Taq-amplified PCR fragments are ligat- ed in just 5 minutes and result in ≥95% recombi-

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Transcription of Tightly Controlled Bacterial Protein Expression - u-szeged.hu

1 PBAD Expression System Tightly Controlled Bacterial Protein Expression The pBAD system offers: Tightly regulated Expression Dose-dependent induction High Protein yields Simplified detection and purification of expressed Protein Controlled Expression for maximum yields Take control of Expression in E. coli with the pBAD Expression System. Tight regulation allows you to turn Expression on or off. Dose-dependent induction lets you easily modulate Expression levels. No other Bacterial Expression system gives you such control. The pBAD Expression System helps you overcome two of In both cases, a Tightly regulated Expression system is the most common problems for heterologous Protein critical for maximizing recombinant Protein yields.

2 Based Expression in bacteria: on the araBAD operon, which controls the arabinose metabolic pathway in E. coli (1,2,3), the pBAD. Toxicity of the recombinant Protein to the host Expression System allows you to precisely modulate lev- Insolubility of the recombinant Protein when it is els to optimize yields. expressed at high, uncontrolled levels Tight regulation The pBAD vectors' unique design give you precise con- Basal Expression levels can be repressed by introducing trol of Expression levels. The araBAD promoter initiates glucose to the growth medium. Glucose acts by lowering gene Expression . It's both positively and negatively regu- cAMP levels, which in turn decreases the binding of lated by the product of the araC gene (4), a transcrip- CAP.

3 As cAMP levels are lowered, transcriptional activa- tional regulator that forms a complex with L-arabinose. tion is decreased. This is ideal when the Protein of inter- In the absence of arabinose, the AraC dimer contacts the est is extremely growth-inhibitive or toxic to the host (3). O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop (Figure 1). For maximum transcription- Figure 1 - Regulation of the araBAD promoter al activation, two events are required: O2. C. Arabinose binds to AraC. The Protein releases the O2. site and binds the I2 site, which is adjacent to the I1 N N. AraC dimer site.

4 This releases the DNA loop and allows transcrip- Pc C No transcription tion to begin (5). I1 I2 pBAD. The cAMP activator Protein (CAP)-cAMP complex L-arabinose binds to the DNA and stimulates binding of AraC to I1. and I2. N. N. Transcription C C. Pc CAP. I1 I2 pBAD. pBAD Expression System Precise control for optimal yields With pBAD, you're in control throughout the entire Figure 2 demonstrates the uniquely tight, arabinose- cell growth and Expression process. Easily optimize dependent induction of Cycle 3 GFP (green fluores- induction and identify the conditions that are best cent Protein ) from pBAD/His.

5 The data show the dis- suited to the unique properties of your Protein . tinctive low levels of uninduced Expression as well as the tight, dose-dependent induction of GFP in the presence of increasing concentrations of arabinose. Figure 2 - Western blot of arabinose induction .0004..0006..0008..0002..002..001..02. % arabinose 0. TOP10 Bacterial cultures containing pBAD/His/GFP were grown to mid-log phase (A600 ~ OD) and induced for 3 hours with increasing doses of ara- binose. One milliliter of cell culture was centrifuged and the pellet resuspend- Cycle 3 GFP ed in 100 l of lysis buffer. Ten microliters of cell lysate was separated by SDS-PAGE and blotted.

6 Protein was detected using the Anti-Xpress Antibody and chemiluminescence. Simplified purification Detecting and purifying proteins expressed from the Control Protein *. With pBAD, it's easy to achieve pBAD vectors is simplified by the presence of epitope maximized Expression , specific Protein detection and tags. Figure 3 demonstrates the simple process of efficient purification. Expression and purification of the 53 kD Positope . Figure 3 - Purification of the Positope Control Protein 1 2 3 4 5 6. The Positope Control Protein was expressed in TOP10 E. coli from the pBAD/Thio-TOPO vector and purified using ProBond Resin.

7 Four micro- grams of total Protein was loaded into each lane of an SDS-PAGE gel. Lane 1: Crude extract Lane 2: Flow through Lane 3: Column wash Lane 4: 100 mM imidazole elution Lane 5: 500 mM imidazole elution Lane 6: Purified, dialyzed Positope Protein * The Positope Control Protein is a recombinant Protein specifically engineered to contain seven different tags for detection with seven different antibodies available from Invitrogen. The Positope Control Protein is intended for use as a positive control for antibody function in western blot experiments. Variety and versatility Invitrogen's pBAD vectors are specifically designed for max- The following features are included in all pBAD vectors: imum Expression and ease of use.

8 Nine pBAD vectors are araBAD promoter for dose-dependent regulation currently available: pBAD102/D-TOPO , pBAD202/D-TOPO , araC gene for tight control of the araBAD promoter pBAD-TOPO , pBAD/Thio-TOPO , pBAD/His, pBAD/Myc- Optimized ribosome binding site for increased transla- His, pBAD-DEST49, pBAD/gIII and pBAD/Thio-E. tion efficiency rrnB transcription termination region for efficient tran- A variety of vector-specific features are available to facili- script processing tate cloning , Protein purification and detection. With so many pBAD vectors to choose from, you're sure to find Read on to find out more about these versatile vectors.

9 One that fits your needs. Directional TOPO cloning : 5 minute cloning , reduced screening The pBAD Directional TOPO Expression Kits offer efficient hang on the TOPO -adapted vector hybridizes to the com- cloning of blunt-ended PCR products generated with proof- plimentary sequence in your PCR product via strand inva- reading DNA polymerases. A choice of ampicillin sion . The topoisomerase I then rapidly performs a ligation (pBAD102/D-TOPO ) or kanamycin (pBAD202/D-TOPO ) that results in greater than 90% of recombinant clones in resistance markers give you the flexibility to design your the correct orientation for Expression .

10 The entire reaction experiments the way you want to. The mechanism for only takes five minutes and maintains directionality, so Directional TOPO cloning is simple (Figure 4). Once the you spend less time screening. PCR product and vector are mixed, a four base pair over- Figure 4 - How Directional TOPO works 1 Design your PCR primer with CACC at 4 Transform E. coli TOPO. Hind III. the 5 end (no modification of the 3 . Pme I. Nco I. Sac I. primer is neccessary). HP thio- CCC TT AAG GGT. redoxin EK Site GGG AAG TGG TTC CCA. V5 epitope 6x His Stop Intermediate Construct 2 Amplify your gene with a proofreading TOPO.


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