Expresso® Biotin Cloning & Expression System

产品说明书

• Ultra-pure protein thru AviTag in vivo biotinylation
• Single column biotin-streptavidin purification.
• Dual affinity tags (AviTag + 6HIS) for full length protein purification
• >90% pure protein in hours with minimal hands on time
• Tight control over expression with Rhamnose promoter
• Optional SUMO tag for increased solubility and on-column cleavage
• Expressioneering™ Technology:overview of Expresso® product line featuring ligase-free cloning

Expresso® Biotin Protein Expression systems offer you the quickest route to biotinylated protein by combining AviTag™ and Expressioneering™ technologies. Simply prepare your insert by PCR and transform it along with the vector, and let the cells do the rest. Go from PCR product to biotinylated protein in 48 hours with minimal hands-on time.

AviTag Expressioneering Generates Biotinylated Protein

Lucigen has partnered with Avidity, LLC to leverage the power of the AviTag™ minimal biotinylation motif and Lucigen's SUMO solubility fusion tag to solve the most difficult problems in protein expression and purification. The included Biotin XCell™ competent cells will efficiently biotinylate protein bearing the AviTag motif in vivo, meaning you can skip the in vitro and chemical methods otherwise required to obtain biotinylated target protein. The SUMO tag will produce large amounts of soluble protein that can be precisely cleaved with its cognate protease. Single column purification of AviTag proteins is completed in a few hours using the biotin-streptavidin interaction, and on column cleavage of the AviTag-SUMO fusion followed by nickel column chromatography of the HIS tagged protease yields >95% pure protein. Other variations of the cloning system allow a variety of dual tag (AviTag + His6) experimental possibilities. Applications of the technology include:

• Protein binding studies
• Rapid protein purification
• Protein detection and ChIP Seq

Figure 1. Expresso Biotin vectors are available for C- or N-terminal biotinylation with optional SUMO tag to further enhance solubility and purification strategies.

• Seamless integration of your target protein with optimized expression elements
• No intervening restriction, protease or recombination sites
• Provides higher yields of perfect protein with no scars or unwanted cloning artifacts

	Lane 1 MW Ladder Lane 2 Uninduced cell lysate Lane 3 Induced cell lysate Lane 4 Induced cell lysate, soluble fraction Lane 5 Resin supernatant (unbound protein) Lane 6 Freed resin-bound target protein
Figure 2. Expression and capture of biotinylated protein. Using a simple autoinduction method, a large amount of protein is expressed and is soluble (lanes 3-4). Due to efficient in vivo biotinylation, most of the target protein is biotinylated and captured using biotin streptavidin purification (lanes 5-6).

Ultra-pure protein easier than ever

Expresso Biotin vectors are also available with the SUMO tag, which increases solubility, protein yield and provides a specific protease site. Using the SUMO N-Biotin version of the vector, you can bind your protein to streptavidin agarose and perform on-column cleavage, leaving you with stringently purified, native target protein.

Figure 3. Workflow diagram of AviTag capture and SUMO Protease on-column cleavage.

	Lane 1 MW Ladder Lane 2 Induced cell lysate (soluble fraction) Lane 3 Lysate after capture of biotinylated target Lane 4 Streptavidin-captured protein Lane 5 Supernatant of untreated resin Lane 6 Streptavidin-captured protein, SUMO Protease treated Lane 7 Supernatant of resin, SUMO Protease treated
Figure 4. Expression and capture of biotinylated protein. A biotinylated protein is expressed and captured on streptavidin agarose (lanes 2-4). Without protease treatment, no protein is freed from the resin (lane 5). Streptavidin agarose was boiled to release all protein, which was then treated with protease to cleave the target (lane 6).

Four Expresso Biotin vector configurations for the ultimate flexibility in fusion tag applications:

Application	N-Biotin	C-Biotin	SUMO N-Biotin	SUMO C-Biotin
Screening / Panning	?	?	?	?
Full-length (dual tag) purification				?
Ultra-pure protein purification			?
Protein Microarrays	?	?	?	?
Chromatin Immunoprecipitation (ChIP Seq, ENCODE elements)	?	?	?	?
Solubility Enhancement			?	?
Protein:Protein Interaction	?	?	?	?
Protein:DNA Interaction	?	?	?	?
Protein:RNA Interaction	?	?	?	?

Figure 5. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR-amplified DNA into the pAviTag vectors. Target genes are amplified with primers that include 18 bases of overlap with the ends of the Expresso vector. The unpurified PCR amplicon is simply mixed with the pAviTag expression plasmids and immediately transformed into the highly competent cells provided. No DNA purification, enzyme treatment, vector preparation, or ligation steps are required

AviTag™ is a trademark of Avidity, LLC. AviTag™ technology (U.S. Patent Nos. 5,723,584, 5,874,239 & 5,932,433) is owned by and licensed from Avidity, LLC, Denver, Colorado.

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The Expresso® Biotin Cloning & Expression System contains pre-processed Vector DNA, single-transformation BIOTIN XCell F’ Chemically Competent Cells (SOLOs), and recovery medium. Also included are BFP positive control insert DNA, transformation positive control pUC DNA, and forward and reverse PCR primers to confirm clones. For protein expression, solutions of Rhamnose, Biotin, Arabinose and Glucose are provided. SUMO kits also contain SUMO Express Protease and cleavage control protein.

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