In vitro Director™ System

cDNA to Protein in Less than a Day . . . No Cloning!

In vitro Director provides a unique ligation mediated PCR method to generate DNA templates for coupled in vitro expression systems such as wheat germ and rabbit reticulocyte. The system utilizes a proprietary blend of ExoClone™ dNTPs that result in controlled directional ligation of Anchor™ sequences to any gene. Anchors provide necessary sequences such as the T7 promoter, Kozak ribosomal binding site, and affinity tags. The combination of In vitro Director with a coupled in vitro expression system and downstream 96-well affinity purification products creates a powerful system for high throughput protein expression and characterization that is unmatched in speed and reliability.

High throughput in vitro expression Universal for any gene Streamlined directional addition of promoters, affinity tags, etc. to PCR amplicons Overcome difficult in vivo expression    – Eukaryotic genes    – Insoluble protein    – Proteins that are toxic to the host cell    – Rapid proteolytic degradation in vivo Rapid protein characterization, protein-protein interaction, labeling and more

Overview of Method: Target Gene to Protein in Less than a Day
Rapid Assembly of Expression Ready DNA Templates
One-Step Coupled In Vitro Protein Synthesis
High-throughput Protein-Protein Interaction Studies
Universal for Plant and Other Eukaryotic Genes
Products

Overview of Method: Target Gene to Protein in Less than a Day

First round PCR to amplify target gene from cDNA or plasmid DNA in the presence of dNTPαS (2-3 hours) Controlled exonuclease III treatment for generation of cohesive ends (10 minutes) Directional ligation of 1st round PCR product to Anchors (30 minutes) Second round PCR to amplify expression ready DNA templates using universal Anchor Primers (2-3 hours) In vitro protein synthesis (1-2 hours)

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Rapid Assembly of Expression Ready DNA Templates

Figure 1 Figure 2

In vitro Director Method Robust, high throughput assembly Validated for >100 genes ranging in size from 239 to 2207 bp No concern of internal cutting sites or rearrangements No cloning, no transformation, no cell culture Excess first round PCR product can be rapidly cloned using Director-Ready™ Vectors Figure 1. Strategy for IκB-α interaction domain mapping experiment. Gene-specific primers containing recommended 5 base overhangs were designed to produce full length IκB-α and 5 truncated versions (serial deletions). Figure 2. Rapid assembly of IκB-α expression ready templates. Lanes 2-7 are the 630 bp-954 bp first round PCR products amplified from an IκB-α-CMV-2 vector using gene-specific primers (see figure 1) and ExoClone™ dNTP mix. After rapid digestion with exonuclease III to produce cohesive ends, purified products were ligated to the 5' c-Myc Anchor™ set. Assembled templates were amplified using universal Anchor Primers to produce expression ready templates (lanes 10-15) containing the T7 promoter, Kozak sequence, c-Myc tag, etc.

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One-Step Coupled In vitro Protein Synthesis

Figure 3. In vitro expression of IκB-α serial deleted fragments. Expression ready templates produced during second round PCR in Figure 2 were used directly in a coupled rabbit reticulocyte expression system. The synthesized c-Myc-IκB-α fragments were purified from the rabbit reticulocyte lysate through a short incubation on Anti-c-Myc 96-well affinity capture plates, eluted and detected on a western blot using monoclonal Anti-c-Myc and a rabbit Anti-mouse-HRP secondary antibody (chemiluminescent detection). The 210-318 amino acid fragments are shown in lanes 1-6 above.

Transcription/Translation of Expression Ready Templates Contain T7 promoters, Kozak consensus ribosomal binding sites, and optional affinity tags through ligation with Anchors 2nd PCR provides enough template for up to 10 transcription/translation reactions Compatible with coupled, uncoupled or linked transcription/translation systems Expression validated in wheat germ and rabbit reticulocyte systems

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High-Throughput Protein-Protein Interaction Studies

Analysis of In Vitro Synthesized Protein Bait/prey proteins for protein-protein interaction studies can be in vitro synthesized or endogenous protein ANTI-FLAG® and Anti-c-Myc 96-well affinity capture plates provide a further streamlined high throughput platform for quantitative analysis MALDI, ELISA or Western blotting may be used for analysis

Figure 4. Binding Domain Mapping of IκB-α. FLAG-p65, expressed in Cos-7 cells, was captured on an ANTI-FLAG 96-well plate. The anchored FLAG-p65 was used to capture interacting proteins from the transcription/translation reactions in Figure 3. Anti-c-Myc-AP/pNPP was used to detect any interacting c-Myc-IκB-α fragments. According to the ELISA results, a necessary domain for interaction with p65 maps to the region between 252 aa and 277 aa of IκB-α, narrowing the area for future studies to a 25 amino acid region. The entire experiment (target DNA amplification to ELISA results) was completed in only 1.5 days.

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Universal for Plant and Other Eukaryotic Genes

Figure 5. Protein-Protein Interaction of Arabidopsis Proteins. Expression Ready templates for Arabidopsis histone-3 (H3) and histone-4 (H4) proteins were prepared using the In vitro Director 5'-c-Myc and 5'-FLAG Anchor Kits. In vitro expression was performed using the Wheat Germ TNT® System. In vitro synthesized c-Myc-H3 and c-Myc H4 (bait proteins) were captured on Anti-c-Myc 96-well plates and incubated with corresponding FLAG-tagged prey proteins (H3 or H4). Protein-protein interaction was detected using ANTI-FLAG-M2-AP conjugate/pNPP. FLAG-BAP was used as a negative control. The left panel shows specific interaction of FLAG-H4 with c-Myc-H3 while the right panel shows specific interaction of FLAG-H3 with c-Myc-H4.

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Products

We also offer a variety of amino acids for production of stable isotope labeled proteins.

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